Monocyte Derived Microvesicles Deliver a Cell Death Message via Encapsulated Caspase-1

Apoptosis depends upon the activation of intracellular caspases which are classically induced by either an intrinsic (mitochondrial based) or extrinsic (cytokine) pathway. However, in the process of explaining how endotoxin activated monocytes are able to induce apoptosis of vascular smooth muscle cells when co-cultured, we uncovered a transcellular apoptosis inducing pathway that utilizes caspase-1 containing microvesicles. Endotoxin stimulated monocytes induce the cell death of VSMCs but this activity is found in 100,000 g pellets of cell free supernatants of these monocytes. This activity is not a direct effect of endotoxin, and is inhibited by the caspase-1 inhibitor YVADcmk but not by inhibitors of Fas-L, IL-1β and IL-18. Importantly, the apoptosis inducing activity co-purifies with 100 nm sized microvesicles as determined by TEM of the pellets. These microvesicles contain caspase-1 and caspase-1 encapsulation is required since disruption of microvesicular integrity destroys the apoptotic activity but not the caspase-1 enzymatic activity. Thus, monocytes are capable of delivering a cell death message which depends upon the release of microvesicles containing functional caspase-1. This transcellular apoptosis induction pathway describes a novel pathway for inflammation induced programmed cell death.     

Direct activation of mitochondrial apoptosis machinery by c-Jun N-terminal kinase in adult cardiac myocytes.

Although oxidative stress causes activation of c-Jun N-terminal kinase (JNK) and apoptosis in many cell types, how the JNK pathway is connected to the apoptosis pathway is unclear. The molecular mechanism of JNK-mediated apoptosis was investigated in adult rat cardiac myocytes in culture as a model system that is sensitive to oxidative stress. Oxidative stress caused JNK activation, cytochrome c release, and apoptosis without new protein synthesis. Oxidative stress-induced apoptosis was abrogated by dominant negative stress-activated protein kinase/extracellular signal-regulated kinase kinase-1 (SEK1)-mediated inhibition of the JNK pathway, whereas activation of the JNK pathway by constitutively active SEK1 was sufficient to cause apoptosis. Inhibition of caspase-9, an apical caspase in the mitochondrial apoptosis pathway, suppressed oxidative stress-induced apoptosis, whereas inhibition of caspase-8 had no effect, indicating that both the JNK pathway and the mitochondrial apoptosis machinery are central to oxidative stress-induced apoptosis. Both JNK and SEK1 localized on mitochondria where JNK was activated by oxidative stress. Furthermore, active JNK caused the release of apoptogenic factors such as cytochrome c from isolated mitochondria in a cell-free assay. These findings indicate that the JNK pathway is a direct activator of mitochondrial death machinery without other cellular components and provide a molecular linkage from oxidative stress to the mitochondrial apoptosis machinery.     

Coxiella burnetii induces apoptosis during early stage infection via a caspase-independent pathway in human monocytic THP-1 cells

The ability of Coxiella burnetii to modulate host cell death may be a critical factor in disease development. In this study, human monocytic THP-1 cells were used to examine the ability of C. burnetii Nine Mile phase II (NMII) to modulate apoptotic signaling. Typical apoptotic cell morphological changes and DNA fragmentation were detected in NMII infected cells at an early stage of infection. FACS analysis using Annexin-V-PI double staining showed the induction of a significant number of apoptotic cells at an early stage of NMII infection. Double staining of apoptotic cell DNA and intracellular C. burnetii indicates that NMII infected cells undergoing apoptosis. Interestingly, caspase-3 was not cleaved in NMII infected cells and the caspase-inhibitor Z-VAD-fmk did not prevent NMII induced apoptosis. Surprisingly, the caspase-3 downstream substrate PARP was cleaved in NMII infected cells. These results suggest that NMII induces apoptosis during an early stage of infection through a caspase-independent pathway in THP-1 cells. In addition, NMII-infected monocytes were unable to prevent exogenous staurosporine-induced apoptotic death. Western blot analysis indicated that NMII infection induced the translocation of AIF from mitochondria into the nucleus. Cytochrome c release and cytosol-to-mitochondrial translocation of the pore-forming protein Bax in NMII infected cells occurred at 24 h post infection. These data suggest that NMII infection induced caspase-independent apoptosis through a mechanism involving cytochrome c release, cytosol-to-mitochondrial translocation of Bax and nuclear translocation of AIF in THP-1 monocytes. Furthermore, NMII infection increased TNF-α production and neutralization of TNF-α in NMII infected cells partially blocked PARP cleavage, suggesting TNF-α may play a role in the upstream signaling involved in NMII induced apoptosis. Antibiotic inhibition of C. burnetii RNA synthesis blocked NMII infection-induced PARP activation. These results suggest that both intracellular C. burnetii replication and secreted TNF-α contribute to NMII infection-triggered apoptosis during an early stage of infection.     

Apo2L/TRAIL is an indirect mediator of apoptosis induced by interferon-alpha in human myeloma cells

Interferon-alpha (IFN-alpha) is currently used for the therapy of multiple myeloma (MM) though it is only effective in some patients. IFN-alpha induces apoptosis in some MM cell lines and it has been proposed to occur through an autocrine loop involving Apo2L/TRAIL. We have analysed the sensitivity to IFN-alpha and Apo2L/TRAIL of five MM cell lines and found no correlation between the apoptosis inducing ability of both cytokines. IFN-alpha-induced apoptosis in MM cells was not prevented by a caspase-8 selective inhibitor (Z-IETD-fmk) or blocking Apo2L/TRAIL. However, human monocytes treated with IFN-alpha release bioactive Apo2L/TRAIL to culture media which was cytotoxic for MM cells resistant to IFN-alpha. We propose that Apo2L/TRAIL released from IFN-alpha-stimulated blood monocytes would be a major mediator of the anti-myeloma effect of IFN-alpha in vivo.     

Mycobacterial infection induces the secretion of high-mobility group box 1 protein

High-mobility group box protein 1 (HMGB1) is a non-histone nuclear protein that acts as a pro-inflammatory cytokine and is released by monocytes and macrophages. Necrotic cells also release HMGB1 at the site of tissue damage which induces a variety of cellular responses, including the expression of pro-inflammatory mediators. This study investigated the secretion of HMGB1 in mycobacterial infection by macrophages in vitro and in the lungs of infected guinea pigs. We observed that infection by mycobacterium effectively induced HMGB1 release in both macrophage and monocytic cell cultures. Culture filtrate proteins from Mycobacterium tuberculosis induced maximum release of HMGB1 compared with different subcellular fractions of mycobacterium. We demonstrated that HMGB1 is released in lungs during infection of M. tuberculosis in guinea pigs and increased HMGB1 secretion in lungs of guinea pigs was delayed by prior vaccination with Mycobacterium bovis BCG. The secretion of cytokines like tumour necrosis factor alpha (TNF-alpha) and Interleukin-1beta was significantly increased when M. bovis BCG-infected cultures of J774A.1 cells were incubated with HMGB1. Among different mycobacterial toll-like receptor ligands, heat-shock protein 65 (HSP65) was found to be more potent in inducing HMGB1 secretion in RAW 264.7 cells. Pharmacological suppression of p38 or extracellular signal-regulated kinase 1/2 mitogen-activated protein kinases with specific inhibitors failed to inhibit HSP65-induced HMGB1 release, but inhibition of c-Jun NH(2)-terminal kinase activation attenuated HMGB1 release. Inhibition of the inducible NO synthase and neutralizing antibodies against TNF-alpha also reduced HMGB1 release stimulated by HSP65. We conclude that HMGB1 is secreted by macrophages during tuberculosis and it may act as a signal of tissue or cellular injury and enhances immune response.     

Mycobacterium tuberculosis infection causes different levels of apoptosis and necrosis in human macrophages and alveolar epithelial cells

Mycobacterium tuberculosis interacts with macrophages and epithelial cells in the alveolar space of the lung, where it is able to invade and replicate in both cell types. M. tuberculosis-associated cytotoxicity to these cells has been well documented, but the mechanisms of host cell death are not well understood. We examined the induction of apoptosis and necrosis of human macrophages (U937) and type II alveolar epithelial cells (A549) by virulent (H37Rv) and attenuated (H37Ra) M. tuberculosis strains. Apoptosis was determined by both enzyme-linked immunosorbent assay (ELISA) and TdT-mediated dUTP nick end labelling (TUNEL) assay, whereas necrosis was evaluated by the release of lactate dehydrogenase (LDH). Both virulent and attenuated M. tuberculosis induced apoptosis in macrophages; however, the attenuated strain resulted in significantly more apoptosis than the virulent strain after 5 days of infection. In contrast, cytotoxicity of alveolar cells was the result of necrosis, but not apoptosis. Although infection with M. tuberculosis strains resulted in apoptosis of 14% of the cells on the monolayer, cell death associated with necrosis was observed in 59% of alveolar epithelial cells after 5 days of infection. Infection with M. tuberculosis suppressed apoptosis of alveolar epithelial cells induced by the kinase inhibitor, staurosporine. Because our findings suggest that M. tuberculosis can modulate the apoptotic response of macrophages and epithelial cells, we carried out an apoptosis pathway-specific cDNA microarray analysis of human macrophages and alveolar epithelial cells. Whereas the inhibitors of apoptosis, bcl-2 and Rb, were upregulated over 2.5-fold in infected (48 h) alveolar epithelial cells, the proapoptotic genes, bad and bax, were downregulated. The opposite was observed when U937 macrophages were infected with M. tuberculosis. Upon infection of alveolar epithelial cells with M. tuberculosis, the generation of apoptosis, as determined by the expression of caspase-1, caspase-3 and caspase-10, was inhibited. Inhibition of replication of intracellular bacteria resulted in an increase in apoptosis in both cell types. Our results showed that the differential induction of apoptosis between macrophages and alveolar epithelial cells represents specific strategies of M. tuberculosis for survival in the host.     

IL-16 is constitutively present in peripheral blood monocytes and spontaneously released during apoptosis

Constitutive expression of the pro-molecule of IL-16 has been found in T cells, mast cells, eosinophils, epithelial cells, fibroblasts, and dendritic cells. Here we show that IL-16 is also constitutively present in >98% of freshly isolated human CD14-positive peripheral blood monocytes when analyzed by flow cytometry. Because pro-IL-16 is cleaved to its bioactive mature form by caspase-3, and caspase-3 is also the pivotal effector of apoptosis in monocytes, we asked whether IL-16 release occurs in monocytes that undergo spontaneous apoptosis. As expected, freshly isolated, unstimulated monocytes underwent spontaneous caspase-3 activation. This apoptosis was paralleled by the loss of intracellular IL-16, as detected by flow cytometry, and the concurrent release of IL-16, as detected by ELISA. In contrast, stimulation with bacterial LPS inhibited caspase-3 activation and significantly inhibited the release of IL-16. As a specificity control, IL-1beta and IL-8 were not released during spontaneous monocyte apoptosis. In summary, our data demonstrate that monocytes contain IL-16 that is released during spontaneous apoptosis.     

Rosmarinic acid induces p56lck-dependent apoptosis in Jurkat and peripheral T cells via mitochondrial pathway independent from Fas/Fas ligand interaction

Apoptosis is one way of controlling immune responses, and a variety of immunosuppressive drugs suppress harmful immune responses by inducing apoptosis of lymphocytes. In this study we observed that rosmarinic acid, a secondary metabolite of herbal plants, induced apoptosis in an p56(lck) (Lck)-dependent manner; Lck(+) Jurkat T cells undergo apoptosis in response to rosmarinic acid (RosA) treatment, whereas Lck(-) Jurkat subclone J.CaM1.6 cells do not. J.CaM1.6 cells with various Lck mutants indicated that Lck SH2 domain, but not Lck kinase activity, was required for RosA-induced apoptosis. RosA induced apoptosis in the absence of a TCR stimulus, and this was not prevented by interruption of the Fas/Fas ligand interaction. Instead, RosA-mediated apoptosis involved a mitochondrial pathway as indicated by cytochrome c release and the complete blockage of apoptosis by an inhibitor of mitochondrial membrane depolarization. Both caspase-3 and -8 were indispensable in RosA-induced apoptosis and work downstream of mitochondria and caspase-9 in the order of caspase-9/caspase-3/caspase-8. In freshly isolated human PBMC, RosA specifically induced apoptosis of Lck(+) subsets such as T and NK cells, but not Lck-deficient cells, including B cells and monocytes. Moreover, RosA's ability to kill T and NK cells was restricted to actively proliferating cells, but not to resting cells. In conclusion, Lck-dependent apoptotic activity may make RosA an attractive therapeutic tool for the treatment of diseases in which T cell apoptosis is beneficial.     

The 19-kDa Mycobacterium tuberculosis protein induces macrophage apoptosis through Toll-like receptor-2

Macrophages infected with Mycobacterium tuberculosis undergo increased rates of apoptosis. Important objectives are to define the microbial factors that cause apoptosis, the mechanisms involved and the impact on infection. The 19-kDa M. tuberculosis glycolipoprotein (p19) is both cell wall-associated and secreted and is a candidate virulence factor. We investigated the potential of recombinant, His-tagged p19 lacking the secretion/acylation signal to induce macrophage apoptosis. The TUNEL assay and annexin V binding to membrane phosphatidylserine were used to measure apoptosis. The results show that p19 does act to induce apoptosis in differentiated THP-1 cells and monocyte-derived macrophages and that this effect is both dose- and time-dependent. Furthermore, this effect of p19 is Toll-like receptor (TLR)-2-mediated because preincubation of either THP-1 cells or TLR-2-expressing CHO cells with anti-TLR-2 mAb inhibited apoptosis induced by p19. Apoptosis of macrophages in response to p19 was found to be caspase-8 dependent and caspase-9 independent consistent with a transmembrane pathway signaling cell death through TLR-2. The viability of M. tuberculosis in cells undergoing apoptosis induced by p19 was significantly reduced suggesting the possibility that this may favor containment of infection. Although native p19 is a mycobacterial glycolipoprotein, based upon the use of recombinant p19 where the acylation signal had been removed, we conclude that it is the polypeptide component of p19 that is responsible for signaling through TLR-2 and that the lipid moiety is not required.     

Apoptosis triggered by Rv1818c, a PE family gene from Mycobacterium tuberculosis is regulated by mitochondrial intermediates in T cells

Ectopic expression of the Mycobacterium tuberculosis PE-family gene Rv1818c, triggers apoptosis in the mammalian Jurkat T cells, which is blocked by anti-apoptotic protein Bcl-2. Although complete overlap is not observed, a considerable proportion of cellular pools of ectopically expressed Rv1818c localizes to mitochondria. However, recombinant Rv1818c does not trigger release of cytochrome c from isolated mitochondria even though Rv1818c protein induced apoptosis of Jurkat T cells. Apoptosis induced by Rv1818c is blocked by the broad-spectrum caspase inhibitory peptide zVAD-FMK. Unexpectedly, Rv1818c-induced apoptosis is not blocked in a Jurkat sub-clone deficient for caspase-8 (JI 9.2) or in cells where caspase-9 function is inhibited or expression of caspase-9 reduced by siRNA, arguing against a central role for these caspases in Rv1818c-induced apoptotic signaling. Depleting cellular pools of the mitochondrial protein Smac/DIABLO substantially reduces apoptosis consistent with mitochondrial involvement in this death pathway. We present evidence that Rv1818c-induced apoptosis is blocked by the co-transfection of an endogenous inhibitor of caspase activation, XIAP in T cells. Additionally, Rv1818c is released into extracellular environment via exosomes secreted by M. tuberculosis infected BM-DC's and macrophages. Furthermore, the extracellular Rv1818c protein can be detected in T cells co-cultured with infected BM-DC's. Taken together, these data suggest that Rv1818c-induced apoptotic signaling is likely regulated in part by the Smac-dependent activation of caspases in T cells.     

Presentation of nitric oxide regulates monocyte survival through effects on caspase-9 and caspase-3 activation

In the absence of survival factors, blood monocytes undergo spontaneous apoptosis, which involves the activation of caspase-3. Although nitric oxide can block caspase-3 activation and promote cell survival, it can also induce apoptosis. We hypothesized that nitrosothiols that promote protein S-nitrosylation would reduce caspase-3 activation and cell survival, whereas nitric oxide donors (such as 1-propamine 3-(2-hydroxy-2-nitroso-1-propylhydrazine (PAPA) NONOate and diethylamine (DEA) NONOate) that do not target thiol residues would not. Using human monocytes as a model, we observed that nitrosothiol donors S-nitrosoglutathione and S-nitroso-N-acetylpenicillamine suppressed caspase-9 and caspase-3 activity and DNA fragmentation. In contrast, PAPA or DEA NONOate did not promote monocyte survival events and appeared to inhibit monocyte survival induced by macrophage colony-stimulating factor. The caspase-3-selective inhibitor DEVD-fluoromethyl ketone reversed DNA fragmentation events, and the caspase-9 inhibitor LEHD-fluoromethyl ketone reversed caspase-3 activity in monocytes treated with PAPA or DEA NONOate in the presence of macrophage colony-stimulating factor. These results were not caused by differences in glutathione levels or the kinetics of nitric oxide release. Moreover, S-nitrosoglutathione and S-nitroso-N-acetylpenicillamine directly blocked the activity of recombinant caspase-3, which was reversed by the reducing agent dithiothreitol, whereas PAPA or DEA NONOate did not block the enzymatic activity of caspase-3. These data support the hypothesis that nitrosylation of protein thiol residues by nitric oxide is critical for promoting the survival of human monocytes.     

Differential regulation of IFN-gamma, TNF-alpha, and IL-10 production by CD4(+) alphabetaTCR+ T cells and vdelta2(+) gammadelta T cells in response to monocytes infected with Mycobacterium tuberculosis-H37Ra.

Mycobacterium tuberculosis bacilli readily activate CD4(+) and gammadelta T cells. CD4(+) and gammadelta T cells were compared for their ability to regulate IFN-gamma, TNF-alpha, and IL-10 production, cytokines with significant roles in the immune response to M. tuberculosis. PBMC from healthy tuberculin positive donors were stimulated with live M. tuberculosis-H37Ra. CD4(+) and gammadelta T cells were purified by negative selection and tested in response to autologous monocytes infected with M. tuberculosis. Both subsets produced equal amounts of secreted IFN-gamma. However, the precursor frequency of IFN-gamma secreting gammadelta T cells was half that of CD4(+) T cells, indicating that gammadelta T cells were more efficient producers of IFN-gamma than CD4(+) T cells. TNF-alpha production was markedly enhanced by addition of CD4(+) and gammadelta T cells to M. tuberculosis infected monocytes, and TNF-alpha was produced by both T cells and monocytes. No differences in TNF-alpha enhancement were noted between CD4(+) and gammadelta T cells. IL-10 production by M. tuberculosis infected monocytes was not modulated by CD4(+) or gammadelta T cells. Thus CD4(+) and gammadelta T cells had similar roles in differential regulation of IFN-gamma, TNF-alpha, and IL-10 secretion in response to M. tuberculosis infected monocytes. However, the interaction between T cells and infected monocytes differed for each cytokine. IFN-gamma production was dependent on antigen presentation and costimulators provided by monocytes. TNF-alpha levels were increased by addition of TNF-alpha produced by T cells and IL-10 production by monocytes was not modulated by CD4(+) or gammadelta T cells.     

Caspase-2 induces apoptosis by releasing proapoptotic proteins from mitochondria

Caspase-2 is one of the earliest identified caspases, but the mechanism of caspase-2-induced apoptosis remains unknown. We show here that caspase-2 engages the mitochondria-dependent apoptotic pathway by inducing the release of cytochrome c (Cyt c) and other mitochondrial apoptogenic factors into the cell cytoplasm. In support of these observations we found that Bcl-2 and Bcl-xL can block caspase-2- and CRADD (caspase and RIP adaptor with death domain)-induced cell death. Unlike caspase-8, which can process all known caspase zymogens directly, caspase-2 is completely inactive toward other caspase zymogens. However, like caspase-8, physiological levels of purified caspase-2 can cleave cytosolic Bid protein, which in turn can trigger the release of Cyt c from isolated mitochondria. Interestingly, caspase-2 can also induce directly the release of Cyt c, AIF (apoptosis-inducing factor), and Smac (second mitochondria-derived activator of caspases protein) from isolated mitochondria independent of Bid or other cytosolic factors. The caspase-2-released Cyt c is sufficient to activate the Apaf-caspase-9 apoptosome in vitro. In combination, our data suggest that caspase-2 is a direct effector of the mitochondrial apoptotic pathway.     

Mycobacterium tuberculosis triggers host type I IFN signaling to regulate IL-1β production in human macrophages

Mycobacterium tuberculosis is a virulent intracellular pathogen that survives in macrophages even in the presence of an intact adaptive immune response. Type I IFNs have been shown to exacerbate tuberculosis in mice and to be associated with disease progression in infected humans. Nevertheless, the mechanisms by which type I IFNs regulate the host response to M. tuberculosis infection are poorly understood. In this study, we show that M. tuberculosis induces an IFN-related gene expression signature in infected primary human macrophages, which is dependent on host type I IFN signaling as well as the mycobacterial virulence factor, region of difference-1. We further demonstrate that type I IFNs selectively limit the production of IL-1β, a critical mediator of immunity to M. tuberculosis. This regulation occurs at the level of IL1B mRNA expression, rather than caspase-1 activation or autocrine IL-1 amplification and appears to be preferentially used by virulent mycobacteria since avirulent M. bovis bacillus Calmette-Guérin (BCG) fails to trigger significant expression of type I IFNs or release of mature IL-1β protein. The latter property is associated with decreased caspase-1-dependent IL-1β maturation in the BCG-infected macrophages. Interestingly, human monocytes in contrast to macrophages produce comparable levels of IL-1β in response to either M. tuberculosis or BCG. Taken together, these findings demonstrate that virulent and avirulent mycobacteria employ distinct pathways for regulating IL-1β production in human macrophages and reveal that in the case of M. tuberculosis infection the induction of type I IFNs is a major mechanism used for this purpose.     

Human immunodeficiency virus type 1 Vpr induces cell cycle arrest at the G(1) phase and apoptosis via disruption of mitochondrial function in rodent cells

Vpr of human immunodeficiency virus type 1 causes cell cycle arrest at the G(2)/M phase and induces apoptosis after G(2)/M arrest in primate cells. We have reported previously that Vpr also induces apoptosis independently of G(2)/M arrest in human HeLa cells. By contrast, Vpr does not induce G(2)/M arrest in rodent cells, but it retards cell growth. To clarify the relationship between cell cycle arrest and apoptosis, we expressed Vpr endogenously in rodent cells and investigated cell cycle profiles and apoptosis. We show here that Vpr induces cell cycle arrest at the G(1) phase and apoptosis in rodent cells. Vpr increased the activity of caspase-3 and caspase-9, but not of caspase-8. Moreover, Vpr-induced apoptosis could be inhibited by inhibitors of caspase-3 and caspase-9, but not by inhibitor of caspase-8. We also showed that Vpr induces the release of cytochrome c from mitochondria into the cytosol and disrupts the mitochondrial transmembrane potential. Finally, we showed that apoptosis occurred in HeLa cells through an identical pathway. These results suggest that disruption of mitochondrial functions by Vpr induces apoptosis via cell cycle arrest at G(1), but that apoptosis is independent of G(2)/M arrest. Furthermore, it appears that Vpr acts species-specifically with respect to induction of cell cycle arrest but not of apoptosis.     

Immunoelectrophoresis of mycobacterial antigens

Polyvalent antiserum to culture filtrate of H37 Ra M. tuberculosis was raised in rabbits. Monospecific antiserum was raised against M. tuberculosis antigen-5, prepared from the culture filtrates by immunoabsorbent affinity chromatography. On immunoelectrophoresis, antigen-5 demonstrated single precipitin arc against polyvalent and monospecific antisera. The culture filtrate antigen demonstrated multiple precipitin arcs against polyvalent antiserum and single precipitin are against monospecific antiserum. Antigen-5 could be isolated and characterized from the culture filtrate of H37 Ra M. tuberculosis. Immunoelectrophoresis could be one of the method to characterize the mycobacterial antigens prepared in the laboratory.     

T cells enhance production of IL-18 by monocytes in response to an intracellular pathogen

We studied the effect of T cells on IL-18 production by human monocytes in response to Mycobacterium tuberculosis. Addition of activated T cells markedly enhanced IL-18 production by monocytes exposed to M. tuberculosis. This effect was mediated by a soluble factor and did not require cell-to-cell contact. The effect of activated T cells was mimicked by recombinant IFN-gamma and was abrogated by neutralizing Abs to IFN-gamma. IFN-gamma also enhanced the capacity of alveolar macrophages to produce IL-18 in response to M. tuberculosis, suggesting that this mechanism also operates in the lung during mycobacterial infection. IFN-gamma increased IL-18 production by increasing cleavage of pro-IL-18 to mature IL-18, as it enhanced caspase-1 activity but did not increase IL-18 mRNA expression. These findings suggest that activated T cells can contribute to the initial immune response by augmenting IL-18 production by monocytes in response to an intracellular pathogen.     

c-Myc and caspase-2 are involved in activating Bax during cytotoxic drug-induced apoptosis

Activation of Bax following diverse cytotoxic stress has been shown to be an essential gateway to mitochondrial dysfunction and activation of the intrinsic apoptotic pathway characterized by cytochrome c release with caspase-9/-3 activation. Interestingly, c-Myc has been reported to promote apoptosis by destabilizing mitochondrial integrity in a Bax-dependent manner. Stress-induced activation of caspase-2 may also induce permeabilization of mitochondria with activation of the intrinsic death pathway. To test whether c-Myc and caspase-2 cooperate to activate Bax and thereby mediate intrinsic apoptosis, small interfering RNA was used to efficiently knock down the expression of c-Myc, caspase-2, and Apaf-1, an activating component in the apoptosome, in two human cancer cell lines, lung adenocarcinoma A-549 and osteosarcoma U2-OS cells. Under conditions when the expression of endogenous c-Myc, caspase-2, or Apaf-1 is reduced 80-90%, cisplatin (or etoposide)-induced apoptosis is significantly decreased. Biochemical studies reveal that the expression of c-Myc and caspase-2 is crucial for cytochrome c release from mitochondria during cytotoxic stress and that Apaf-1 is only required following cytochrome c release to activate caspases-9/-3. Although knockdown of c-Myc or caspase-2 does not affect Bax expression, caspase-2 is important for cytosolic Bax to integrate into the outer mitochondrial membrane, and c-Myc is critical for oligomerization of Bax once integrated into the membrane.