Cigarette smoke prevents apoptosis through inhibition of caspase activation and induces necrosis

Emphysema is characterized by enlargement of the distal airspaces in the lungs due to destruction of alveolar walls. Alveolar endothelial and epithelial cell apoptosis induced by cigarette smoke is thought to be a possible mechanism for this cell loss. In contrast, our studies show that cigarette smoke condensate (CSC) induces necrosis in alveolar epithelial cells and human umbilical vein endothelial cells. Furthermore, study of the cell death pathway in a model system using Jurkat cells revealed that in addition to inducing necrosis, CSC inhibited apoptosis induced by staurosporine or Fas ligation, with both effects prevented by the antioxidants glutathione and dithiothreitol. Time course experiments revealed that CSC inhibited an early step in the caspase cascade, whereby caspase-3 was not activated. Moreover, cell-free reconstitution of the apoptosome in cytoplasmic extracts from CSC-treated cells, by addition of cytochrome-c and dATP, did not result in activation of caspases-3 or -9. Thus, smoke treatment may alter the levels of pro- and antiapoptogenic factors downstream of the mitochondria to inhibit active apoptosome formation. Therefore, unlike previous studies, cell death in response to cigarette smoke by necrosis and not apoptosis may be responsible for the loss of alveolar walls and inflammation observed in emphysema.     

Induction of apoptosis in Vero cells by Newcastle disease virus requires viral replication, de-novo protein synthesis and caspase activation

Newcastle disease virus causes (NDV) apoptotic death of infected cells. In the present study, the stimulus that provoked the induction of apoptosis in infected cells was examined. Vero cells infected with NDV developed apoptosis as characterized by DNA fragmentation and decreased DNA content. In presence of ammonium chloride, infected cells did not show reduced DNA content indicating the requirement of virus entry for the induction of apoptosis. UV-inactivated NDV did not induce apoptosis in cells suggesting the need of virus replication. Although cycloheximide blocked NDV-induced apoptosis, actinomycin-D did not, suggesting that de-novo viral protein synthesis was critical for the induction of apoptosis. In addition, activation of caspases was also detected by flowcytometry, indirect fluorescent and colorimetric assays. Based on the results, it was concluded that NDV-induced apoptosis in Vero cells required virus replication, de-novo protein synthesis and caspase activation.     

Granzyme A loading induces rapid cytolysis and a novel form of DNA damage independently of caspase activation.

Cytotoxic lymphocytes trigger apoptosis by releasing perforin and granzymes (Grn). GrnB activates the caspase apoptotic pathway, but little is known about GrnA-induced cell death. Perforin was used to load recombinant GrnA and GrnB and enzymatically inactive variants into target cells. GrnA induces single-strand DNA breaks that can be labeled with Klenow polymerase and visualized on alkaline gels. GrnA-induced DNA damage but not cytolysis requires GrnA proteolysis. GrnA-induced membrane perturbation, nuclear condensation, and DNA damage are unimpaired by caspase blockade. GrnA fails to induce cleavage of caspase-3, lamin B, rho-GTPase, or PARP. GrnA-induced cytotoxicity and cleavage of PHAP II, a previously identified GrnA substrate, are unimpaired in Jurkat cells that overexpress bcl-2. Therefore, GrnA activates a novel apoptotic pathway.     

The membrane attack complex of complement induces caspase activation and apoptosis

Activation of the terminal pathway of the complement system leads to insertion of terminal complement complexes (C5b-9) into the cell membrane, which may induce cytolysis. Recent data indicate that the terminal complement pathway can also result in apoptosis in vivo. To further define the cell death pathway induced by complement, we examined induction of apoptosis by complement in vitro. Rat mesangial cells opsonized with a complement-activating antibody and exposed to rat serum as a complement source underwent apoptotic cell death in a time- and dose-dependent fashion, as demonstrated by membrane exposure of phosphatidylserine and fragmentation of nuclei. No significant apoptosis was detected in either cultures treated with C6-deficient serum or in control cultures. The pan-caspase-inhibitor zVAD-fmk inhibited complement-induced apoptosis completely. In line with this observation, complement induced cleavage and activation of caspase 3. Importantly, cellular exposure to purified cytolytically inactive C5b-9, in the absence of antibody and early complement components, also resulted into caspase activation and apoptosis. Together, these results indicate that C5b-9 is involved in induction of apoptosis via a caspase-dependent pathway. Apoptosis as a consequence of complement-mediated cell damage may provide an explanation for the presence of apoptosis in inflammatory processes, for instance in hyperacute xenograft rejection.     

MYC‐driven malignant transformation of mature murine B cells requires inhibition of both intrinsic apoptosis and p53 activity

Increased expression of the oncogene MYC is a common feature of many B‐cell malignancies, however MYC overexpression by itself is not sufficient for transformation, and additional genetic events are required, although the exact nature of these remains unknown. In patients and in transgenic mouse models, oncogenic transformation may occur in B cells at various differentiation stages interacting with complex microenvironments. B‐cell oncogenesis often occurs after prolonged periods of time, making it difficult to accurately identify the genetic events required for transformation. An in vitro system, where malignant transformation of primary B cells could be analyzed, would facilitate the identification of genetic events required for transformation. Here, we describe such a system and show that primary murine B cells rapidly become transformed upon forced expression of MYC, in conjunction with simultaneous inhibition of the ARF/p53 axis via overexpression of BMI1, as well as through downregulation of p19^ARF or expression of a dominant‐negative p53 and suppression of intrinsic apoptosis through overexpression of BCLXL or MCL1. Established tumor cells remained addicted to expression of the lymphoma‐inducing genes. In mice, transformed cells rapidly established fatal B‐cell lymphomas. Our results suggest that transformation of normal mature B cells into lymphomas can occur as a consequence of three defined events.     

Hydrogen sulfide prevents apoptosis of human PMN via inhibition of p38 and caspase 3

Hydrogen sulfide, together with carbon monoxide and nitric oxide, is now considered a gasotransmitter able to induce specific cellular responses. As hydrogen sulfide is a component of several natural compounds known to be effective in many inflammatory pathologies, particularly of the respiratory tract, we studied its effects in vitro on the survival and bactericidal activity of purified human neutrophils. We found that (1) HS(-) ions promote the survival of granulocytes, but not that of lymphocytes or eosinophils, cultured in serum-free medium; (2) the pro-survival effect of HS(-) is due to inhibition of caspase-3 cleavage and p38 MAP kinase phosphorylation; (3) the bactericidal activity of neutrophils is not impaired by hydrogen sulfide. We conclude that HS(-) promotes the short-term survival of neutrophils potentially accelerating the resolution of inflammatory processes and preventing the occurrence of new ones.     

Clostridium perfringens iota-toxin requires activation of both binding and enzymatic components for cytopathic activity

Iota-toxin is produced by Clostridium perfringens type E strains and consists of two independent components, the enzymatic and binding components, referred to as Ia and Ib, respectively. A recombinant C. perfringens strain, strain 667/pMRP147, produced processed Ia and partially processed Ib, while a recombinant C. perfringens type A strain, strain TS133/pMRP147, in which the VirR-VirS two-component system is inactivated, produced only precursor forms of Ia and Ib. This suggests that iota-toxin is processed by a VirR-VirS-responsive protease, although not completely in the recombinant type A strain. The precursor forms of Ia and Ib were purified from cultures of the latter strain, and their proteolytic activation was examined. Treatment with proteases cleaved off small peptides (9 to 13 amino acid residues) and a 20-kDa peptide from the N termini of the Ia and Ib precursors, respectively, leading to their active forms. They were activated efficiently by alpha-chymotrypsin, pepsin, proteinase K, subtilisin, and thermolysin but only weakly by trypsin, as demonstrated by the cell-rounding assay. lambda-Protease from the C. perfringens type E strain, which was found to be a zinc-dependent protease related to thermolysin, activated iota-toxin as efficiently as did alpha-chymotrypsin. These results suggest that lambda-protease is most responsible for the activation of iota-toxin in type E strains.     

Membrane receptor-mediated apoptosis and caspase activation in the differentiated EoL-1 eosinophilic cell line

Caspases are key molecules in the control of apoptosis, but relatively little is known about their contribution to eosinophil apoptosis. We examined caspase-3, -8, and -9 activities in receptor ligation-dependent apoptosis induction in the differentiated human eosinophilic cell line EoL-1. Differentiated EoL-1 exhibited bi-lobed nuclei, eosinophil-associated membrane receptors, and basic granule proteins. Annexin-V fluorescein isothiocyanate binding to EoL-1 revealed significant (P<0.01) apoptosis induction in cells cultured for 20 h with monoclonal antibodies (mAb) specific for CD45 (71%+/-4.3), CD45RA (58%+/-2.3), CD45RB (68%+/-2.4), CD95 (47%+/-2.6), and CD69 (52%+/-2.1) compared with control (23%+/-1.6) or CD45RO mAb (27%+/-3.9). The pan-caspase inhibitor Z-Val-Ala-Asp-fluoromethylketone (fmk) and inhibitors of caspase-8 (Z-Ile-Glu-Thr-Asp-fmk) and caspase-9 (Z-Leu-Glu-His-Asp-fmk) significantly inhibited mAb-induced apoptosis of EoL-1 but had no effect on constitutive (baseline) apoptosis at 16 and 20 h. Caspase activity was analyzed using the novel CaspaTag trade mark technique and flow cytometry. EoL-1 treated with pan-CD45, CD45RA, CD45RB, and CD95 mAb exhibited caspase-3 and -9 activation at 12 h post-treatment, which increased at 16 and 20 h. Activated caspase-8 was detected 12 and 16 h after ligation with CD45, CD45RA, CD45RB, and CD95 mAb followed by a trend toward basal levels at 20 h. CD69 ligation resulted in caspase-3 activation, a modest but significant activation of caspase-8, and a loss in mitochondrial transmembrane potential but had no significant effect on activation of caspase-9. Thus, the intrinsic and extrinsic caspase pathways are involved in controlling receptor ligation-mediated apoptosis induction in human eosinophils, findings that may aid the development of a more targeted, anti-inflammatory therapy for asthma.     

Caspase activation by granzyme B is indirect,and caspase autoprocessing requires the release of proapoptotic mitochondrial factors

Apoptosis in response to granzyme B involves activation of caspase-dependent target cell death pathways. Herein, we show that granzyme B initiates caspase processing but cannot fully process procaspase-3 in intact Jurkat T leukemia or NT2 neuronal cells. Rather, the release from mitochondria of proapoptotic mediators cytochrome c, Smac/Diablo, and HtrA2/Omi facilitates full activation of caspases that results from autoprocessing. Bcl-2 overexpression in mitochondria suppresses the release of these proapoptotic molecules, resulting in cell survival despite partial procaspase processing by granzyme B. We propose that binding of inhibitor of apoptosis (IAP) proteins to partially processed procaspases inhibits cell death unless mitochondrial disruption also occurs in response to granzyme B or activated BH3-domain proteins such as truncated Bid.     

Mercury-induced apoptosis in human lymphocytes: caspase activation is linked to redox status

There is growing evidence that heavy metals, in general, and mercurial compounds, in particular, are toxic to the human immune system. We have previously shown that methyl mercuric chloride (MeHgCl) is a potent human T-cell apoptogen; moreover, mitochondria appear to be a target organelle for the induction of cell death. The objective of this study was to determine the impact of MeHgCl on mitochondrial function in lymphocytes in terms of modulating reactive oxygen species (ROS) generation, thiol status, and caspase activation. Using the fluorescent probe, 3,3'-dihexyloxacarbocyanine, we demonstrated that exposure to MeHgCl for 1 h resulted in a profound decrease in the mitochondrial transmembrane potential. We next observed the release of cytochrome c from mitochondria into the cytosol; significant translocation was noted between 4 and 8 h following treatment with mercury. ROS generation was monitored by following the conversion of dihydroethidium to the fluorescent product, ethidium. Kinetic analysis indicated that ROS generation was maximal after 16 h of exposure to MeHgCl. The toxicant also depleted the thiol reserves of the cell; glutathione levels were depleted in a dose-dependent fashion reaching minimal levels at 16 h. Real-time RT-PCR analysis demonstrated a significant reduction in both glutathione S-transferase and glutathione peroxidase gene expression in mercury-treated cells. Finally, after 16 h of treatment with MeHgCl, we observed activation of caspase-8, -9, and -3 along with increased expression of caspase-8 and -9. We propose that the target organelle for MeHgCl is the mitochondrion and that induction of oxidative stress is critical to activation of death-signaling pathways. Additonally, mercury acts as a genotoxin significantly altering the expression of genes that affect cell survival and apoptosis.     

Psychosine-induced apoptosis in a mouse oligodendrocyte progenitor cell line is mediated by caspase activation

Imidacloprid is an insecticide which has the nicotinic acetylcholine receptors (nAChRs) as its primary site of action; acetylcholine is the major excitatory neurotransmitter in the insect central nervous system (CNS). In this study, the action of imidacloprid was tested using the synapses of the respiratory central pattern generator of the beetle Tenebrio molitor. The no observed effect concentration (NOEC) for imidacloprid was estimated to be between 0.001 and 0.010 microM. A concentration of 0.10 microM caused hyperexcitation in firing of the respiratory motoneurons, while the concentration of 1.00 microM caused an abrupt increase in their frequency and then a complete inhibition of the activity of the respiratory motoneurons. The possible implication of the action of such low concentrations of imidacloprid in the contraction of the respiratory muscles is also demonstrated and discussed.     

The role of calpain in caspase activation during etoposide induced apoptosis in T cells.

T cells treated with the drug etoposide undergo apoptotic death characterized by early evidence of nuclear damage followed by loss of mitochondrial integrity and cell lysis. Calpains and caspases are cytoplasmic proteases and there is increasing evidence of cross-talk between these proteases in death pathways. In this study we have investigated the role of calpain, in etoposide-triggered apoptosis in the 2B4 murine T cell hybridoma. Cell permeable inhibitors of calpain, ALLnM, E64 and calpeptin that block Fas ligand-Fas-mediated death in T cells, blocked etoposide-induced nuclear damage, loss of mitochondrial integrity and cell lysis. A broad spectrum peptide inhibitor of caspases, ZVAD-fmk, partially blocked nuclear damage but poorly inhibited mitochondrial damage or cell lysis triggered by etoposide. Etoposide-induced expression of the cleaved, proteolytically active form of caspase 3, and DEVD-ase activity, detected prior to nuclear damage, were blocked in the presence of calpain inhibitors. Collectively, these data describe a role for calpain in regulating etoposide-induced apoptosis via a caspase-dependent pathway in T cells.     

Activation of cAMP signaling enhances Fas-mediated apoptosis and activation-induced cell death through potentiation of caspase 8 activation

Defects in Fas receptor signaling lead to compromised maintenance of lymphocyte homeostasis and peripheral immune tolerance, leading in turn to autoimmune disorders. Therefore, agents that can enhance Fas-mediated apoptosis may be therapeutically useful in management of such disorders. In this study, we focused on the effect of cAMP on Fas-mediated apoptosis in human T cells. We show that elevation of intracellular cAMP levels by forskolin, an activator of adenylyl cyclase, 3-isobutyl-1-methylxanthine, an inhibitor of cyclic nucleotide phosphodiesterases, or prostaglandin E(2) potentiates Fas-induced apoptosis in Jurkat cells. Accordingly, cAMP was found to enhance the cleavage of caspase 8 at death-inducing signaling complex and lead to augmentation of the processing of Fas effector proteins. We also demonstrate that cAMP enahnaces Fas-induced apoptosis in normal human T cells and activation-induced cell death in Jurkat cells. These findings provide a rationale for investigating the feasibility of using cAMP-elevating agents to potentiate apoptosis in T cells with aberrant Fas signaling.     

Full activation of the T cell receptor requires both clustering and conformational changes at CD3

T cell receptor (TCR-CD3) triggering involves both receptor clustering and conformational changes at the cytoplasmic tails of the CD3 subunits. The mechanism by which TCRalphabeta ligand binding confers conformational changes to CD3 is unknown. By using well-defined ligands, we showed that induction of the conformational change requires both multivalent engagement and the mobility restriction of the TCR-CD3 imposed by the plasma membrane. The conformational change is elicited by cooperative rearrangements of two TCR-CD3 complexes and does not require accompanying changes in the structure of the TCRalphabeta ectodomains. This conformational change at CD3 reverts upon ligand dissociation and is required for T cell activation. Thus, our permissive geometry model provides a molecular mechanism that rationalizes how the information of ligand binding to TCRalphabeta is transmitted to the CD3 subunits and to the intracellular signaling machinery.     

Tim-4 inhibition of T-cell activation and T helper type 17 differentiation requires both the immunoglobulin V and mucin domains and occurs via the mitogen-activated protein kinase pathway

Emerging experimental data suggest an important role for the T-cell immunoglobulin mucin 1 (Tim-1):Tim-4 pathway in autoimmune and alloimmune responses in vivo. Using a Tim-4 ectodomain human IgG Fc fusion protein we studied the role of Tim-4 in T-cell activation, signalling and differentiation responses in vitro. We demonstrate that Tim-4Fc can inhibit naive and pre-activated T-cell activation, proliferation and cytokine secretion via a Tim-1-independent pathway. Tim-4 contains immunoglobulin variable (IgV) and mucin domains; to identify which domain accounts for the inhibitory effect novel Tim-4 fusion proteins containing either the IgV or mucin domain were generated. We demonstrate that both IgV and mucin domains are required for the inhibitory effects and that they are mediated at least in part by inhibition of extracellular signal-regulated kinase pathway activity. Given the emerging interest in the role of the Tim family in T helper type 17 (Th17) cells, which play an important role in autoimmune disease and transplantation tolerance, our data show that Tim-4Fc can prevent polarization of CD4(+) T cells to the Th17 phenotype. Collectively, our results highlight an inhibitory role for Tim-4Fc in vitro, which we propose is mediated by a receptor other than Tim-1. In addition, this study provides new insights into the role of Tim-4Fc in regulating Th17 immune responses and may open a new avenue for autoimmune therapy.     

Induction of apoptosis of human macrophages in vitro by Legionella longbeachae through activation of the caspase pathway

The cytotoxicity of the facultative intracellular bacterium, Legionella longbeachae, an important cause of legionellosis, was characterised. Apoptosis was induced in HL-60 cells, a human macrophage-like cell line, during the early stages of infection and induction of apoptosis correlated with cytotoxicity. Apoptosis was confirmed by agarose gel electrophoresis of fragmented DNA, surface exposure of phosphatidylserine and propidium iodide labelling of host cell nuclei. The involvement of macrophage infectivity potentiator (Mip) protein, a known virulence factor of L. longbeachae, was also examined. A mip mutant of L. longbeachae induced apoptosis of HL-60 cells but failed to multiply intracellularly, suggesting that intracellular replication of L. longbeachae is not essential for the induction of apoptosis of HL-60 cells. Furthermore, induction of apoptosis of L. longbeachae-infected macrophages was mediated by activation of the caspase pathway but might be independent of tumour necrosis factor-alpha- and Fas-mediated signal transduction pathways.     

Visilizumab induces apoptosis of mucosal T lymphocytes in ulcerative colitis through activation of caspase 3 and 8 dependent pathways

Visilizumab, a humanized low-Fc receptor binding anti-CD3 antibody, induces rapid clinical response in patients with steroid-refractory ulcerative colitis (UC). Several effective treatments in IBD have been linked to the induction of mucosal T cell apoptosis. The aim of the present study was to evaluate the effect of visilizumab on the apoptosis of lamina propria (LP) and peripheral blood (PB) lymphocytes isolated from patients with UC. Visilizumab induced dose- and time-dependent apoptosis of LP T cells isolated from non-IBD individuals, UC or CD patients. Maximal effect was seen at a concentration of 100 ng/ml and it was 33% for normal, 34% for UC and 23% for CD LP T cells following 24 h stimulation. Visilizumab induced apoptosis predominantly of CD4⁺ LP T cells, whereas CD8⁺ LP T cells were relatively resistant to apoptosis. Visilizumab did not induce apoptosis of PB T cells from UC patients. Visilizumab-induced apoptosis of LP T cells was dependent on caspase 3 and 8, but not caspase 9 activation and did not involve the Fas/FasL pathway. Low-Fc receptor binding Abs such as visilizumab may be highly effective for the treatment of UC through induction of apoptosis of LP T cells and rapid elimination of lesional pathogenic T cells in the gut mucosa.     

Mechanism of triptolide-induced apoptosis: effect on caspase activation and Bid cleavage and essentiality of the hydroxyl group of triptolide

Triptolide is a compound extracted from the Chinese herb Tripterygium wilfordii Hook. f. Triptolide has potent anticancer activity. However, the mechanisms by which triptolide exerts its anticancer activities remain unclear. To explore the molecular mechanisms involved in the anticancer activity of triptolide, we have examined the effect of triptolide on the growth of pancreatic carcinoma PANC-1 and cervical adenocarcinoma HeLa cells. We found that treatment of both HeLa and PANC-1 cells with triptolide potently suppressed cell growth and induced apoptosis, indicated by nuclear fragmentation and blebbing. In both HeLa and PANC-1 cells, apoptosis induced by triptolide was associated with activation of both caspase-3 and caspase-8, and cleavage of poly(ADP-ribose) polymerase and Bid. Moreover, in HeLa cells, caspase-9 is also significantly activated in response to triptolide. Overexpression of Bcl-2 in HeLa cells substantially attenuated triptolide-induced apoptosis. Interestingly, substitution of the 14-OH of triptolide with an acetyl group abrogated both its anticancer and its antiinflammatory activities. Our studies suggest that triptolide may exert its anticancer effects by initiating apoptosis through both death-receptor- and mitochondria-mediated pathways. Our results indicate that both the apoptosis-promoting and the antiinflammatory activities of triptolide depend on the 14-OH group.     

Aspartic protease and caspase 3/7 activation are central for macrophage apoptosis following infection with Escherichia coli

Macrophages are vital for host defense against microbial infections. We have previously shown that infection of macrophages with a nonpathogenic strain of Escherichia coli induces apoptosis rapidly. Here, we demonstrate that infection of macrophages results in the activation of caspases prior to the induction of the intrinsic apoptosis pathway. Caspases 9 and 3 are activated prior to the release of intermembrane mitochondrial protein cytochrome C into the cytosol in infected macrophages. Treatment with an inhibitor to caspase 9 has no effect on the death of macrophages and does not prevent activation of the downstream effector caspase 3/7. In contrast, an inhibitor to caspase 3/7 reduces cell death in E. coli-infected macrophages. Although caspase 9 is not required, activation of aspartic proteases, of which cathepsin D is one of the central members, is essential for activation of caspase 3/7. Treatment with pepstatin A, an inhibitor of aspartic proteases, markedly diminishes the activation of cathepsin D and caspase 3/7 and reduces death in E. coli-infected macrophages. Collectively, these data suggest that cathepsin D activation of caspase 3/7 may be required for inducing one of the death pathways elicited by E. coli.