Granzyme B: a natural born killer

Summary: A main pathway used by cytotoxic T lymphocytes (CTLs) and natural killer cells to eliminate pathogenic cells is via exocytosis of granule components in the direction of the target cell, delivering a lethal hit of cytolytic molecules. Amongst these, granzyme B and perforin have been shown to induce CTL‐mediated target cell DNA fragmentation and apoptosis. Once released from the CTL, granzyme B binds its receptor, the mannose‐6‐phosphate/insulin‐like growth factor II receptor, and is endocytosed but remains arrested in endocytic vesicles until released by perforin. Once in the cytosol, granzyme B targets caspase‐3 directly or indirectly through the mitochondria, initiating the caspase cascade to DNA fragmentation and apoptosis. Caspase activity is required for apoptosis to occur; however, in the absence of caspase activity, granzyme B can still initiate mitochondrial events via the cleavage of Bid. Recent work shows that granzyme B‐mediated release of apoptotic factors from the mitochondria is essential for the full activation of caspase‐3. Thus, granzyme B acts at multiple points to initiate the death of the offending cell. Studies of the granzyme B death receptor and internal signaling pathways may lead to critical advances in cell transplantation and cancer therapy.     

Granzyme B leakage-induced cell death: a new type of activation-induced natural killer cell death

Activation-induced natural killer (NK) cell death is very rapid compared to activation-induced T or B cell death. Here we show that NK cell activation is accompanied by the leakage of granzymeB from intracellular granules into the cytoplasm. Evidence for granzyme B leakage includes the formation of granzyme B/serine proteinase inhibitor 9 (PI-9) complexes that are detected by immunoprecipitation as well as colocalization of granzyme B and PI-9 detected by immunocytochemistry. The pro-apoptotic molecule Bid, a specific substrate for granzyme B, was cleaved within 2 min following CD2-induced NK cell activation, suggesting that granzyme B triggers apoptosis by directing Bid to mitochondrial membranes. The granzyme B/PI-9 protein ratio was found to mirror the percentage of CD2-induced NK cell death, suggesting that an excess of leaked granzyme B over its inhibitor is a major determinant of cell death. We suggest that granzyme B leakage-induced cell death is an important determinant of activation-induced NK cell death and that this process may be important for the fate of NK cells which encounter malignant or virus-infected cells.     

Granzyme B-induced apoptosis requires both direct caspase activation and relief of caspase inhibition

Cytotoxic lymphocytes employ Granzyme B as a potent initiator of apoptosis to cleave and activate effector caspases. Unexpectedly, cells transfected with Bcl-2 were resistant to granzyme B-induced killing, suggesting that a mitochondrial pathway was critical. Utilizing cells expressing a dominant-negative caspase 9, the current study demonstrated that caspase activation via the apoptosome was not required. Indeed, cleavage of caspase 3 to p20 still occurred in Bcl-2-transfectants but processing to p17 was blocked. This blockade was recapitulated by the Inhibitor-of-Apoptosis-Protein XIAP and relieved by Smac/DIABLO. Thus granzyme B mediates direct cleavage of caspase 3 and also activates mitochondrial disruption, resulting in the release of proapoptotic proteins that suppress caspase inhibition. Engagement of both pathways is critical for granzyme-induced killing.     

Cytolytic cells induce HMGB1 release from melanoma cell lines

High mobility group box 1 (HMGB1) is one of the recently defined damage-associated molecular pattern molecules, passively released from necrotic cells and secreted by activated macrophage/monocytes. Whether cytolytic cells induce HMGB1 release from tumor cells is not known. We developed a highly sensitive method for detecting intracellular HMGB1 in tumor cells, allowing analysis of the type of cell death and in particular, necrosis. We induced melanoma cell death with cytolytic lymphokine-activated killing (LAK) cells, tumor-specific cytolytic T lymphocytes, TRAIL, or granzyme B delivery and assessed intracellular HMGB1 retention or release to investigate the mechanism of HMGB1 release by cytolytic cells. HMGB1 release from melanoma cells (451Lu, WM9) was detected within 4 h and 24 h following incubation with IL-2-activated PBMC (LAK activity). HLA-A2 and MART1 or gp100-specific cytolytic T lymphocytes induced HMGB1 release from HLA-A2-positive and MART1-positive melanoma cells (FEM X) or T2 cell-loaded, gp100-specific peptides. TRAIL treatment, however, induced HMGB1 release, and it is interesting that this extrinsic pathway-mediated cell death was blocked with the pancaspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone. Conversely, granzyme B delivery did not induce HMGB1 release. HMGB1, along with other intracellular factors released from tumor cells induced by cytolysis, may be important components of the disordered tumor microenvironment. This has important implications for the immunotherapy of patients with cancer. Specifically, HMGB1 may promote healing or immune reactivity, depending on the nature of the local inflammatory response and the presence (or absence) of immune effectors.     

Apoptosis induction by interleukin-2-activated cytotoxic lymphocytes in a squamous cell carcinoma cell line and Daudi cells - involvement of reactive oxygen species-dependent cytochrome c and reactive oxygen species-independent apoptosis-inducing factors

Investigation of the induction of apoptosis by cytotoxic lymphocytes has mainly focused on the signalling associated with Fas and its adaptor proteins. The signal pathway via mitochondria, however, has not been sufficiently elucidated in cytotoxic lymphocyte-induced apoptosis. We examined the release of mitochondrial proapoptotic factors by lymphokine-activated killer (LAK) cells in two cell lines. LAK cell-induced DNA fragmentation of the target cells was suppressed to approximately 50% of control levels by the addition of neutralizing monoclonal antibody to Fas and a granzyme B inhibitor. When intracellular reactive oxygen species (ROS) were scavenged, the LAK cell-induced DNA fragmentation was decreased to approximately 60% of the non-treated cell level. Co-cultivation of Daudi cells with LAK cells increased cytosolic and mitochondrial ROS levels. Activation of procaspase-3 and apoptosis by treatment of oral squamous cell carcinoma cells (OSC) with LAK cells was partially inhibited by pretreatment of OSC cells with ROS scavengers and mitochondrial complex inhibitors. Furthermore, cytochrome c and apoptosis-inducing factor (AIF) were released from mitochondria by OSC cell treatment with supernatants of LAK cells. The supernatant-induced cytochrome c release was suppressed by mitochondrial complex inhibitors, but the inhibitors did not inhibit the release of AIF. These results indicate that LAK cells induce target cell apoptosis via not only the Fas/Fas ligand system and granzyme B, but also ROS-dependent cytochrome c and ROS-independent AIF release.     

Coxiella burnetii inhibits activation of host cell apoptosis through a mechanism that involves preventing cytochrome c release from mitochondria

Coxiella burnetii is an obligate intracellular pathogen and the etiological agent of the human disease Q fever. C. burnetii infects mammalian cells and then remodels the membrane-bound compartment in which it resides into a unique lysosome-derived organelle that supports bacterial multiplication. To gain insight into the mechanisms by which C. burnetii is able to multiply intracellularly, we examined the ability of host cells to respond to signals that normally induce apoptosis. Our data show that mammalian cells infected with C. burnetii are resistant to apoptosis induced by staurosporine and UV light. C. burnetii infection prevented caspase 3/7 activation and limited fragmentation of the host cell nucleus in response to agonists that induce apoptosis. Inhibition of bacterial protein synthesis reduced the antiapoptotic effect that C. burnetii exerted on infected host cells. Inhibition of apoptosis in C. burnetii-infected cells did not correlate with the degradation of proapoptotic BH3-only proteins involved in activation of the intrinsic cell death pathway; however, cytochrome c release from mitochondria was diminished in cells infected with C. burnetii upon induction of apoptosis. These data indicate that C. burnetii can interfere with the intrinsic cell death pathway during infection by producing proteins that either directly or indirectly prevent release of cytochrome c from mitochondria. It is likely that inhibition of apoptosis by C. burnetii represents an important virulence property that allows this obligate intracellular pathogen to maintain host cell viability despite inducing stress that would normally activate the intrinsic death pathway.     

TRAIL-induced apoptosis requires Bax-dependent mitochondrial release of Smac/DIABLO.

Recent reports suggest that a cross-talk exists between apoptosis pathways mediated by mitochondria and cell death receptors. In the present study, we report that mitochondrial events are required for apoptosis induced by the cell death ligand TRAIL (TNF-related apoptosis-inducing ligand) in human cancer cells. We show that the Bax null cancer cells are resistant to TRAIL-induced apoptosis. Bax deficiency has no effect on TRAIL-induced caspase-8 activation and subsequent cleavage of Bid; however, it results in an incomplete caspase-3 processing because of inhibition by XIAP. Release of Smac/DIABLO from mitochondria through the TRAIL-caspase-8-tBid-Bax cascade is required to remove the inhibitory effect of XIAP and allow apoptosis to proceed. Inhibition of caspase-9 activity has no effect on TRAIL-induced caspase-3 activation and cell death, whereas expression of the active form of Smac/DIABLO in the cytosol is sufficient to reconstitute TRAIL sensitivity in Bax-deficient cells. Our results show for the first time that Bax-dependent release of Smac/DIABLO, not cytochrome c, from mitochondria mediates the contribution of the mitochondrial pathway to death receptor-mediated apoptosis.     

Possible key role of granzyme B in keratoacanthoma regression

It is still controversial whether keratoacanthoma is to be considered as a well differentiated variant of squamous cell-carcinoma or a separate entity. As opposed to malignant potential of squamous cell-carcinoma, keratoacanthoma is characterized by a spontaneous regression. However, in some cases, otherwise typical keratoacanthoma can behave aggressively showing the signs of perineural and perivascular invasion and metastases in regional lymph nodes. The most important feature that separates these two closely related entities is a tendency of keratoacanthoma to regress. Causes and detailed mechanism of this regression are still not completely elucidated. Within the past few years, it has become evident that the molecular events regulating cell survival and apoptosis are important contributors to the overall kinetics of benign and malignant cell growth. Immunological mechanisms have been implicated in a phenomenon of spontaneous tumor regression. Recent studies suggested that the tumor regression is dependent mainly on the immune response mediated by cytotoxic T lymphocytes (CD8+), together with helper T cells (CD4+). Cytotoxic T lymphocytes can kill tumor cells and mediate tumor regression in vivo through two distinct molecular mechanisms: Fas/Fas ligand and granzyme B/perforin mediated pathways. Tumor cells are capable of developing different escape mechanisms in order to overcome their sensitivity to apoptotic signals. However, granzyme B, contained in cytolytic granules released upon target cell recognition, can also cause tumor cell death and consequently tumor regression by direct damage to non-nuclear structures through a caspase-independent pathway. Therefore, we propose a key role of plasticity in the granzyme B mediated cell death pathway in the killing of changed tumor cells, resulting in keratoacanthoma regression through apoptosis or direct damage of tumor cells. On the other hand, insufficient activation of cytotoxic T lymphocytes and decreased release or activity of granzyme B could be responsible for squamous cell-carcinoma progression and occasional aggressive behavior in keratoacanthomas. As a first step in confirming or refuting our hypothesis, we suggest a thorough immunohistochemical study of the presence of granzyme B and its activity in keratoacanthoma and squamous cell-carcinoma samples. To our knowledge, no such study has been performed so far.     

Processing of the Nedd2 precursor by ICE-like proteases and granzyme B

Background : The Nedd2/Ich-1 protein belongs to a growing family of mammalian cysteine proteases similar to interleukin-1β converting enzyme (ICE). Because of their similarity to the Caenorhabditis elegans cell death protein CED-3, the ICE-like proteins are thought to play a key role in the execution of apoptosis. The active form of ICE is a tetramer consisting of two heterodimers (p20 + p10)₂ derived from the cleavage of the pro-enzyme.
Results : In the present communication we show that the p51 Nedd2 precursor (pro-Nedd2) is also cleaved into p20-like (p19) and p10-like (p12) subunits by extracts prepared from cultured cell lines. Extracts from apoptotic NIH-3T3 cells but not normal growing NIH-3T3 cells also contained pro-Nedd2 cleaving activity. The processing of pro-Nedd2 by cell extracts was inhibited by characteristic inhibitors of ICE-like proteases. Additionally we show that pro-Nedd2 (p51) can be processed in vitro by active CPP32 and ICE, and to a lesser extent by Mch2 and Nedd2. Granzyme B, a serine protease required for cytotoxic T lymphocyte (CTL) mediated killing of target cells, also cleaved pro-Nedd2 to p19 + p12 subunits.
Conclusions : Our observations suggest that Nedd2 activation requires cleavage by one or more ICE-like proteases that lie upstream in the proteolytic cascade. Cleavage of pro-Nedd2 by granzyme B indicates that Nedd2 may be one of the downstream effectors in the CTL-mediated killing of target cells.     

Macrophages express granzyme B in the lesion areas of atherosclerosis and rheumatoid arthritis

Granzyme B is a major mediator of the cytotoxic immune response by inducing target cell death when internalized in the presence of perforin. Recently, several studies have focused on another role of granzyme B, which is extracellular matrix (ECM) remodeling through the degradation of ECM proteins. In order to investigate the expression pattern of granzyme B in the lesion areas of atherosclerosis and rheumatoid arthritis, we performed immunohistochemistry and in situ hybridization analyses using human atherosclerotic plaques and the synovial tissues of rheumatoid arthritic- and osteoarthritic-joints. In atherosclerotic plaques, granzyme B was expressed by macrophages in areas such as the boundary regions between media and intima, areas around necrotic cores, and in shoulder regions. In the synovial tissues of rheumatoid arthritic-joints, the expression of granzyme B was strongly observed in the lining layers where the majority of cells are macrophages and also in perivascular areas where macrophages and a small number of lymphocytes were mixed to form diffuse cellular aggregates. Granzyme B-positive cells were not detected in osteoarthritic synovium. Furthermore, the expression of granzyme B has been induced in the human macrophage cell line, THP-1, by ECM proteins or agents which induce macrophage differentiation. These observations indicate that macrophages should be added to the list of cell types that express granzyme B in human inflammatory diseases and that granzyme B may play a role in macrophage functions that are associated with disease progression.     

Bcl-2 and Bcl-xL overexpression inhibits cytochrome c release,activation of multiple caspases,and virus release following coxsackievirus B3 infection

Coxsackievirus B3, a cytopathic virus in the family Picornaviridae, induces degenerative changes in host cell morphology. Here we demonstrate cytochrome c release and caspases-2, -3, -6, -7, -8, and -9 processing. Enforced Bcl-2 and Bcl-xL expression markedly reduced release of cytochrome c, presentation of the mitochondrial epitope 7A6, and depressed caspase activation following infection. In comparison, cell death using TRAIL ligand caused caspase-8 processing prior to cytochrome c release and executioner caspases and cell death was only partially rescued by Bcl-2 and Bcl-xL overexpression. Disruption of the mitochondrial inner membrane potential following CVB3 infection was not inhibited by zVAD.fmk treatment. Bcl-2 or Bcl-xL overexpression or zVAD.fmk treatment delayed the loss of host cell viability and decreased progeny virus release following infection. Our data suggest that mitochondrial release of cytochrome c may be an important early event in caspase activation in CVB3 infection, and, as such, may contribute to the loss of host-cell viability and progeny virus release.     

Cleavage of caspase family members by granzyme B: a comparative study in vitro

The aspartase granzyme B is one of the major components of the granules involved in cell killing by cytotoxic T lymphocytes. Granzyme B has been shown to activate the apoptotic death pathway in the target cell, and this involves activation of members of the caspase (CASP) protein family. Therefore, activational cleavage of mouse (m) CASP proforms by granzyme B was examined in vitro. CASP can be subdivided in the CASP-1 (interleukin-1β-converting enzyme; ICE) subfamily, the CASP-2 (Ich1) subfamily, and the CASP-3 (CPP32) subfamily. Our results reveal that the proforms of the CASP-3 subfamily members mCASP-3 and mCASP-7 are hydrolyzed by granzyme B, while proforms of CASP-2 and CASP-1 subfamily members are not directly cleaved. Only one CASP-3 subfamily member, pro-mCASP-6, was not proteolytically cleaved by granzyme B. These results indicate that two members of the CASP-3 subfamily, but no others, become activated by granzyme B.     

Differential involvement of p38 MAP kinase pathway and Bax translocation in the mitochondria-mediated cell death in TCR- and dexamethasone-stimulated thymocytes

Mitochondria play a central role in many apoptotic reactions. Although mitochondrial apoptotic changes and caspase activation have been demonstrated in the apoptotic thymocytes, cell death signal through mitochondria in TCR-stimulated thymocytes has not been fully understood. In this study, we show that TCR stimulation induced disruption of mitochondrial transmembrane potential (Delta Psi(m)), the cytochrome c release from mitochondira, capase-3 activation, and the cell death of thymocytes. Bongkrekic acid, an inhibitor of Delta Psi(m) disruption, blocked the cytochrome c release from mitochondria and the following caspase-3-mediated cell death. Furthermore, a pro-apoptotic Bcl-2 family protein, Bax, but not Bad or Bid, was translocated from cytosol to mitochondria in TCR-stimulated thymocytes. This translocation and the following apoptotic changes were inhibited by SB203580, a p38 kinase inhibitor, in a specific manner. These results suggest that activated p38 kinase pathway by TCR stimulation induces translocation of Bax to mitochondria, causing Delta Psi(m) disruption, and the release of cytochrome c, which finally induces caspase-3-mediated apoptosis in thymocytes.     

Analysis of apoptotic and antiapoptotic signalling pathways induced by Helicobacter pylori.

BACKGROUND AND AIMS: Although it is reported that Helicobacter pylori induces apoptosis on gastric epithelial cells, the mechanism remains unknown. Antiapoptotic effects generated by H pylori have not yet been evaluated. METHODS: (1) H pylori strains (type 1 wild, TN2-deltacagE, TN2-deltavacA) were cocultured with MKN45, TMK1, and HeLa cells, and cell viability and apoptosis were assessed by trypan blue exclusion and DNA laddering, respectively. (2) Activation of caspases-3, 7, and 8, cytochrome c release from the mitochondria, and Fas, Fas associated death domain protein (FADD), Bax, Bak, and Bcl-X expression were evaluated by immunoblot analysis. (3) To investigate whether nuclear factor kappa B (NFkappaB) activation induced by cag pathogenicity island (PAI) positive H pylori affects antiapoptosis, MKN45 cells stably expressing super-repressor IkappaBalpha were cocultured with H pylori, and cell viability and caspase activation were evaluated. NFkappaB regulated gene expression was also evaluated by ribonuclease protection assay. RESULTS: (1) Wild-type and deltavacA mutant H pylori induced apoptosis more potently than the deltacagE mutant. Inhibition of cell contact between H pylori and cancer cells and heat killing H pylori diminished cell death. (2) Caspases-3, 7, and 8 were activated time dependently by H pylori as well as by the agonist anti-Fas. Cytochrome c release from mitochondria was observed and was not inhibited by caspase-8 inhibitor. Although protein expression of Fas, FADD, Bax, Bak, and Bcl-X in the whole cell lysates was not changed by H pylori, Bax was decreased from mitochondria free cytosol suggesting that Bax was translocated into mitochondria. (3) Cell death and the activities of caspases-3 and 8 were promoted in MKN45 cells stably expressing super-repressor IkappaBalpha that inhibits NFkappaB activation. Antiapoptotic proteins c-IAP1 and c-IAP2 were upregulated by the wild-type strains. CONCLUSION: cag PAI positive H pylori is capable of inducing apoptotic effects mainly through the mitochondrial pathway. Antiapoptotic effects mediated by NFkappaB activation were also observed.     

Et-DHA通过激活线粒体途径和T细胞granzyme B诱导HepG2细胞凋亡

 目的：本研究探讨了二十二碳六烯酸乙酯（Et-DHA）对人肝癌HepG2细胞凋亡的影响。方法：HepG2细胞用于检测Et-DHA的抑癌活性,MTT法检测Et-DHA对HepG2细胞的直接抑制作用,Hoechst 33258荧光染色观察细胞的形态特征,ELISA法检测Et-DHA处理后HepG2细胞的活性氧簇（ROS）释放量、总超氧化物歧化酶（SOD）和caspase-9活性,Western blotting法检测胞质和线粒体中Bax、Bak、Bid、Bcl-2、Smac和细胞色素C(Cyt C),以及胞质中cleaved caspase-8、cleaved caspase-9和cleaved caspase-3的水平;T细胞与HepG2细胞共培养,进一步观察Et-DHA处理后T细胞的增殖对HepG2细胞活性的影响,并检测了颗粒酶（granzyme）B的水平。结果：Et-DHA显著抑制HepG2细胞的生长（P＜0.05）,这种抑制作用具浓度效应和时程效应;Et-DHA处理后HepG2细胞的ROS释放量增加,但总SOD活性无明显变化,caspase-9活性显著上升（P＜0.05）;线粒体上的促凋亡蛋白Bax、Bak和Bid水平增加,而抑凋亡蛋白Bcl-2以及线粒体中Cyt C和Smac的水平降低,胞质中的Cyt C、Smac、cleaved caspase-8、cleaved caspase-9、cleaved caspase-3以及cleaved Bid水平呈剂量性升高。另外 T细胞和HepG2细胞共培养组在Et-DHA的诱导下,HepG2细胞的凋亡程度与Et-DHA单独作用时相比进一步增加。在Et-DHA刺激下,T细胞内granzyme B上调,释放到HepG2 细胞内的granzyme B明显增多。结论:Et-DHA可能主要通过线粒体内源性途径以及caspase-8途径,激活caspase-3,诱导HepG2细胞凋亡,以及通过间接活化T细胞,促使 granzyme B增多,从而增强对HepG2细胞的毒性作用。     

Human mast cells produce and release the cytotoxic lymphocyte associated protease granzyme B upon activation

Mast cells are widely distributed throughout the body and express effector functions in allergic reactions, inflammatory diseases, and host defense. Activation of mast cells results in exocytosis of preformed chemical mediators and leads to novel synthesis and secretion of lipid mediators and cytokines. Here, we show that human mast cells also express and release the cytotoxic lymphocyte-associated protease, granzyme B. Granzyme B was active and localized in cytoplasmic granules, morphologically resembling those present in cytotoxic lymphocytes. Expression and release of granzyme B by mast cell-lines HMC-1 and LAD 2 and by cord blood- and mature skin-derived human mast cells depended on the mode of activation of these cells. In mast cell lines and cord blood-derived mast cells, granzyme B expression was mainly induced by non-physiological stimuli (A23187/PMA, Compound 48/80) and substance P. In contrast, mature skin-derived mast cells only produced granzyme B upon IgE-dependent stimulation. We conclude that granzyme B is expressed and released by human mast cells upon physiologic stimulation. This suggests a role for granzyme B as a novel mediator in mast cell biology.     

Embryonic neuronal death due to neurotrophin and neurotransmitter deprivation occurs independent of Apaf-1

Apoptotic protease-activating factor-1 (Apaf-1), dATP, and procaspase-9 form a multimeric complex that triggers programmed cell death through the activation of caspases upon release of cytochrome c from the mitochondria into the cytosol. Although cell death pathways exist that can bypass the requirement for cytochrome c release and caspase activation, several gene knockout studies have shown that the cytochrome c-mediated apoptotic pathway is critical for neural development. Specifically, the number of neuronal progenitor cells is abnormally increased in Apaf-1-, caspase-9-, caspase-3-deficient mice. However, the role of the cytochrome c cell death pathway for apoptosis of postmitotic, differentiated neurons in the developing brain has not been investigated in vivo. In this study we investigated embryonic neuronal cell death caused by trophic factor deprivation or lack of neurotransmitter release by analyzing Apaf-1/tyrosine kinase receptor A (TrkA) and Apaf-1/Munc-18 double mutant mice. Histological analysis of the double mutants' brains (including cell counting and terminal (TdT)-mediated dUTP-biotin nick end labeling (TUNEL) staining) reveals that neuronal cell death caused by these stimuli can proceed independent of Apaf-1.We propose that a switch between apoptotic programs (and their respective proteins) characterizes the transition of a neuronal precursor cell from the progenitor pool to the postmitotic population of differentiated neurons.     

Functional Significance of Adhesion Molecules in Fas-Dependent Apoptotic Cell Death Induced by Interleukin-2-Activated T Cells

We investigated the functional significance of the adhesion molecules CD2 and lymphocyte function-associated antigen-1 (LFA-1: CD11a/CD18) in Fas-Fas ligand (FasL) death pathway. Interleukin-2-activated T cells expressed a large amount of FasL protein and could efficiently kill a Fas-sensitive leukemic cell line, MML-1. The major part (over 80%) of MML-1 cell death was Fas-dependent. Antibodies to CD2 and CD11a/CD18 completely inhibited MML-1 target cell lysis, whereas effector to target cell binding was partially reduced or not affected at all. These results suggest that effector/target interaction via CD2/CD58 and LFA-1/CD54 systems would be essential for triggering target cell death. More interestingly, there is the discordance in the ability of anti-CD2, and particularly anti-LFA-1 antibodies, to block Fas-dependent cell death versus effector to target conjugate formation. This suggests some non-adhesive role for CD2 and LFA-1 in induction of Fas-dependent cell death. Although these antibodies were capable of inhibiting T cell proliferative response, there was no significant reduction of FasL or granzyme B expression. Thus, the signaling pathway for growth inhibition via CD2 and LFA-1 could not be linked to signaling for FasL and granzyme B expression.     

Bid, a critical mediator for apoptosis induced by the activation of Fas/TNF-R1 death receptors in hepatocytes

The Bcl-2 family proteins consist of both antiapoptosis and pro-apoptosis members that regulate apoptosis typically at the mitochondrial level, mainly by controlling the release of cytochrome c and other mitochondrial apoptotic events. However, death signals mediated by Fas/TNF-R1 receptors can usually activate caspases directly, bypassing the need for mitochondria and escaping the regulation by Bcl-2 family proteins. Bid is a novel pro-apoptosis Bcl-2 family protein that is activated by Caspase 8 in response to Fas/TNF-R1 death receptor activation. Activated Bid is translocated to mitochondria and induces cytochrome c release, which in turn activates the downstream caspases. This Bid-mediated pathway is critical in hepatocyte apoptosis induced by Fas/TNF-R1 engagement, where direct activation of cytosolic caspase cascade seems inefficient. The dependence on Bid, and thus on the mitochondrial cytochrome c release, of hepatocyte apoptosis induced by the death receptors also renders it sensitive to the inhibitory regulation by the anti-apoptosis members of the Bcl-2 family proteins, such as Bcl-2 and Bcl-xL. Moreover, the revealing of this death pathway in hepatocytes is important to the understanding of the pathogenesis of a number of hepatic diseases such as hepatitis or endotoxemia-related hepatic failure.