Granzyme B is dispensable in the development of diabetes in non-obese diabetic mice

Pancreatic beta cell destruction in type 1 diabetes is mediated by cytotoxic CD8(+) T lymphoctyes (CTL). Granzyme B is an effector molecule used by CTL to kill target cells. We previously showed that granzyme B-deficient allogeneic CTL inefficiently killed pancreatic islets in vitro. We generated granzyme B-deficient non-obese diabetic (NOD) mice to test whether granzyme B is an important effector molecule in spontaneous type 1 diabetes. Granzyme B-deficient islet antigen-specific CD8(+) T cells had impaired homing into islets of young mice. Insulitis was reduced in granzyme B-deficient mice at 70 days of age (insulitis score 0.043±0.019 in granzyme B-deficient versus 0.139±0.034 in wild-type NOD mice p<0.05), but was similar to wild-type at 100 and 150 days of age. We observed a reduced frequency of CD3(+)CD8(+) T cells in the islets and peripheral lymphoid tissues of granzyme B-deficient mice (p<0.005 and p<0.0001 respectively), but there was no difference in cell proportions in the thymus. Antigen-specific CTL developed normally in granzyme B-deficient mice, and were able to kill NOD islet target cells as efficiently as wild-type CTL in vitro. The incidence of spontaneous diabetes in granzyme B-deficient mice was the same as wild-type NOD mice. We observed a delayed onset of diabetes in granzyme B-deficient CD8-dependent NOD8.3 mice (median onset 102.5 days in granzyme B-deficient versus 57.50 days in wild-type NOD8.3 mice), which may be due to the delayed onset of insulitis or inefficient priming at an earlier age in this accelerated model of diabetes. Our data indicate that granzyme B is dispensable for beta cell destruction in type 1 diabetes, but is required for efficient early activation of CTL.     

The granzyme B inhibitor, protease inhibitor 9, is mainly expressed by dendritic cells and at immune-privileged sites

Granzyme B is released from CTLs and NK cells and an important mediator of CTL/NK-induced apoptosis in target cells. The human intracellular serpin proteinase inhibitor (PI)9 is the only human protein able to inhibit the activity of granzyme B. As a first step to elucidate the physiological role of PI9, PI9 protein expression in various human tissues was studied. A mAb directed against human PI9 was developed, which specifically stained PI9-transfected COS-7 cells, and was used for immunohistochemistry. Both in primary lymphoid organs and in inflammatory infiltrates, PI9 was present in different subsets of dendritic cells. Also T-lymphocytes in primary and organ-associated lymphoid tissues were PI9 positive. Endothelial cells of small vessels in most organs tested as well as the endothelial layer of large veins and arteries showed strong PI9 staining. Surprisingly, high PI9 protein expression was also found at immune-privileged sites like the placenta, the testis, the ovary, and the eye. These data fit with the hypothesis that PI9 is expressed at sites where degranulation of CTL or NK cells is potentially deleterious.     

Molecular ordering of the caspase activation cascade initiated by the cytotoxic T lymphocyte/natural killer (CTL/NK) protease granzyme B

Granzyme B is a major cytotoxic T lymphocyte/natural killer (CTL/NK) granule protease that can activate members of the caspase family of cysteine proteases through processing of caspase zymogens. However, the molecular order and relative importance of caspase activation events that occur in target cells during granzyme B-initiated apoptosis has not been established. Here, we have examined the hierarchy of granzyme B-initiated caspase activation events using a cell-free system where all caspases are present at physiological levels. We show that granzyme B initiates a two-tiered caspase activation cascade involving seven caspases, where caspase-3 is required for the second tier of caspase activation events. Using a two-dimensional gel-based proteomics approach we have also examined the scale of granzyme B-initiated alterations to the proteome in the presence or absence of effector caspase-3 or -7. These studies indicate that granzyme B targets a highly restricted range of substrates and orchestrates cellular demolition largely through activation of caspase-3.     

Granzyme B short-circuits the need for caspase 8 activity during granule-mediated cytotoxic T-lymphocyte killing by directly cleaving Bid

Cytotoxic T lymphocytes (CTL) can trigger an apoptotic signal through the Fas receptor or by the exocytosis of granzyme B and perforin. Caspase activation is an important component of both pathways. Granzyme B, a serine proteinase contained in granules, has been shown to proteolytically process and activate members of the caspase family in vitro. In order to gain an understanding of the contributions of caspases 8 and 3 during granule-induced apoptosis in intact cells, we have used target cells that either stably express the rabbitpox virus-encoded caspase inhibitor SPI-2 or are devoid of caspase 3. The overexpression of SPI-2 in target cells significantly inhibited DNA fragmentation, phosphatidylserine externalization, and mitochondrial disruption during Fas-mediated cell death. In contrast, SPI-2 expression in target cells provided no protection against granzyme-mediated apoptosis, mitochondrial collapse, or cytolysis, leading us to conclude that SPI-2-inhibited caspases are not an essential requirement for the granzyme pathway. Caspase 3-deficient MCF-7 cells were found to be resistant to CTL-mediated DNA fragmentation but not to CTL-mediated cytolysis and loss of the mitochondrial inner membrane potential. Furthermore, we demonstrate that granzyme B directly cleaves the proapoptotic molecule Bid, bypassing the need for caspase 8 activation of Bid. These results provide evidence for a two-pronged strategy for mediating target cell destruction and provide evidence of a direct link between granzyme B activity, Bid cleavage, and caspase 3 activation in whole cells.     

A Colorimetric Assay that Specifically Measures Granzyme B Proteolytic Activity: Hydrolysis of Boc-Ala-Ala-Asp-S-Bzl

The serine protease Granzyme B (GzmB) mediates target cell apoptosis when released by cytotoxic T lymphocytes (CTL) or natural killer (NK) cells. GzmB is the most studied granzyme in humans and mice and therefore, researchers need specific and reliable tools to study its function and role in pathophysiology. This especially necessitates assays that do not recognize proteases such as caspases or other granzymes that are structurally or functionally related. Here, we apply GzmB’s preference for cleavage after aspartic acid residues in a colorimetric assay using the peptide thioester Boc-Ala-Ala-Asp-S-Bzl. GzmB is the only mammalian serine protease capable of cleaving this substrate. The substrate is cleaved with similar efficiency by human, mouse and rat GzmB, a property not shared by other commercially available peptide substrates, even some that are advertised as being suitable for this purpose. This protocol is demonstrated using unfractionated lysates from activated NK cells or CTL and is also suitable for recombinant proteases generated in a variety of prokaryotic and eukaryotic systems, provided the correct controls are used. This assay is a highly specific method to ascertain the potential pro-apoptotic activity of cytotoxic molecules in mammalian lymphocytes, and of their recombinant counterparts expressed by a variety of methodologies.     

The granzyme B inhibitor, PI-9, is present in endothelial and mesothelial cells, suggesting that it protects bystander cells during immune responses

Proteinase inhibitor 9 (PI-9) is a 42-kDa human intracellular serpin present in cytotoxic lymphocytes (CLs). PI-9 is an extremely efficient inhibitor of the pro-apoptotic CL granule proteinase granzyme B and is thought to function in the cytosol of CLs to protect against apoptosis induced by endogenously expressed or released granzyme B, particularly during target cell killing. Here we show by immunohistochemistry that PI-9 is also present in endothelial cells, in every tissue examined. Cultured endothelial cells express functional PI-9 (as assessed by binding to recombinant granzyme B) localized to the cytoplasm and nucleus. Immunohistochemistry also showed PI-9 in mesothelial cells, and this was confirmed by analysis of primary cells cultured from pleural and serous effusions. Granzyme B expression was not detected in either endothelial or mesothelial cells. In both cell types, PI-9 is up-regulated at the mRNA and protein level by exposure to the phorbol ester PMA, consistent with a response to inflammatory stimuli. We postulate that PI-9 is present in these lining cell types to protect against misdirected, free granzyme B released during a local immune response.     

Mechanism of defective natural killer cell activity in patients with AIDS is associated with defective distribution of tubulin

Previous studies have demonstrated the importance of some cytoskeleton components in killing mechanisms. In fact, a microtubule and microfilament (MF) rearrangement in the lytic sequence of CTL and NK cells has been observed. In particular, MF seem to be related to the binding phase, because MF inhibitors suppress the binding of NK cells to the target, whereas microtubule inhibitors suppress only the killing phase. In this paper, the distribution of two cytoskeleton components, actin and alpha- and beta-tubulin, has been studied in PBL from AIDS patients, who maintain the capacity to bind to the target cell line K562 but are not able to kill it. PBL were labeled with mAb to these two cytoskeleton components, and then indirect immunofluorescence was used to visualize their distribution in the conjugates. A normal polarization of actin in the effector PBL was found, whereas no tubulin rearrangement was evident in the effector and target cells. On the contrary, in conjugates of PBL or large granular lymphocytes from normal donors and K562, a polarization of actin in the effector cell and a polarization of tubulin both in the effector and in the target cells, at the site of the attachment, was evident. These data suggest that a deficiency of tubulin rearrangement may underlie the inability of the NK cells from AIDS patients to kill their target.     

Mannose 6-phosphate/insulin-like growth factor II receptor is a death receptor for granzyme B during cytotoxic T cell-induced apoptosis

The serine proteinase granzyme B is crucial for the rapid induction of target cell apoptosis by cytotoxic T cells. Granzyme B was recently demonstrated to enter cells in a perforin-independent manner, thus predicting the existence of a cell surface receptor(s). We now present evidence that this receptor is the cation-independent mannose 6-phosphate/insulin-like growth factor receptor (CI-MPR). Inhibition of the granzyme B-CI-MPR interaction prevented granzyme B cell surface binding, uptake, and the induction of apoptosis. Significantly, expression of the CI-MPR was essential for cytotoxic T cell-mediated apoptosis of target cells in vitro and for the rejection of allogeneic cells in vivo. These results suggest a novel target for immunotherapy and a potential mechanism used by tumors for immune evasion.     

Nuclear translocation of granzyme B in target cell apoptosis

Granzyme B is the prototypic member of a family of serine proteases localized to the cytolytic granules of cytotoxic lymphocytes. Together with another granule protein, perforin, granzyme B is capable of inducing all aspects of apoptotic death in target cells. A number of granzyme B substrates have been identified and it has been demonstrated that granzyme B is responsible, directly or indirectly, for the morphological nuclear changes observed in target cell apoptosis, including DNA fragmentation. In an earlier study, we showed that granzyme B binds to a nuclear protein in a manner dependent on its enzymatic activity. Here, we demonstrate that granzyme B is translocated rapidly to the nucleus in cells that have been induced to undergo apoptosis by a granzyme-dependent process, and that translocation is dependent on caspase activity. Appearance of granzyme B in the nucleus of target cells precedes the detection of DNA fragmentation. Although not directly responsible for DNA fragmentation, these data suggest a nuclear role for granzyme B in target cell apoptosis. c-Abl nuclear functions.     

Granzyme B proteolyzes receptors important to proliferation and survival, tipping the balance toward apoptosis

Granzyme B is critical to the ability of natural killer cells and cytotoxic T lymphocytes to induce efficient cell death of virally infected or tumor cell targets. Although granzyme B can cleave and activate caspases to induce apoptosis, granzyme B can also cause caspase-independent cell death. Thirteen prospective granzyme B substrates were identified from a cDNA expression-cleavage screen, including Hsp70, Notch1, fibroblast growth factor receptor-1 (FGFR1), poly-A-binding protein, cAbl, heterogeneous nuclear ribonucleoprotein H', Br140, and intersectin-1. Validation revealed that Notch1 is a substrate of both granzyme B and caspases, whereas FGFR1 is a caspase-independent substrate of granzyme B. Proteolysis of FGFR1 in prostate cancer cells has functionally relevant consequences that indicate its cleavage may be advantageous for granzyme B to kill prostate cancer cells. Therefore, granzyme B not only activates pro-death functions within a target, but also has a previously unidentified role in inactivating pro-growth signals to cause cell death.     

Perforin is recaptured by natural killer cells following target cells stimulation for cytotoxicity

When encountering target cells, NK (natural killer) cells exocytose Pfn (perforin) and granzyme B to kill challengers. We previously reported that granzyme B is recycled and reused by NK cells via clathrin-dependent endocytosis. However, whether Pfn, a main secretory vesicle content, indispensible to granzyme B killing, undergoes endocytosis remains unknown. We demonstrate that Pfn is recaptured by early endosomes of NK cells via a clathrin-dependent endocytosis after target cell stimulation. Inhibition of clathrin-dependent endocytosis significantly attenuated the cytotoxicity of NK cells. The data suggest that the recovery of Pfn contributes to the cytotoxicity of NK cells. The assay of endocytosis of lytic molecule presents a particular focus for exploring the mechanism of abnormal cytotoxicity of NK cells.     

Localization of Fas ligand in cytoplasmic granules of CD8+ cytotoxic T lymphocytes and natural killer cells: participation of Fas ligand in granule exocytosis model of cytotoxicity

Fas ligand (FasL) has been implicated in cytotoxic T lymphocyte (CTL)- and natural killer (NK) cell-mediated cytotoxicity. In the present study, we investigated the localization of FasL in murine CTL and NK cells. Immunocytochemical staining showed that FasL was stored in cytoplasmic granules of CD8+ CTL clones and in vivo activated CTL and NK cells, where perforin and granzyme A also resided. Immunoelectron microscopy revealed that FasL was localized on outer membrane of the cytoplasmic granules, while perforin was localized in internal vesicles. Western blot analysis showed that the membrane-type FasL of 40 kDa was stored in CD8+ CTL clones but not in CD4+ CTL clones. By utilizing a granule exocytosis inhibitor (TN16), we demonstrated that FasL translocated onto cell surface upon degranulation of anti-CD3-stimulated CD8+ CTL clones. Moreover, TN16 markedly inhibited the FasL-mediated cytotoxicity by CD8+ T cell clones and NK cells. These results suggested a substantial contribution of FasL to granule exocytosis-mediated target cell lysis by CD8+ CTL and NK cells.     

A central role for Bid in granzyme B-induced apoptosis

Granzyme B, a protease released from cytotoxic lymphocytes, has been proposed to induce target cell death by cleaving and activating the pro-apoptotic Bcl-2 family member Bid. It has also been proposed that granzyme B can induce target cell death by activating caspases directly, by cleaving caspase substrates, and/or by cleaving several non-caspase substrates. The relative importance of Bid in granzyme B-induced cell death has therefore remained unclear. Here we report that cells isolated from various tissues of Bid-deficient mice were resistant to granzyme B-induced cell death. Consistent with the proposed role of Bid in regulating mitochondrial outer membrane permeabilization, cytochrome c remained in the mitochondria of Bid-deficient cells treated with granzyme B. Unlike wild type cells, Bid-deficient cells survived and were then able to proliferate normally, demonstrating the critical role for Bid in mediating granzyme B-induced apoptosis.     

Granzyme B cleavage of fibronectin disrupts endothelial cell adhesion,migration and capillary tube formation

Dysregulated angiogenesis contributes to the pathogenesis of chronic inflammatory diseases. Modulation of the extracellular matrix by immune-derived proteases can alter endothelial cell–matrix interactions as well as endothelial cell sprouting, migration and capillary formation. Granzyme B is a serine protease that is expressed by a variety of immune cells, and accumulates in the extracellular milieu in many chronic inflammatory disorders that are associated with dysregulated angiogenesis. Although granzyme B is known to cleave fibronectin, an essential glycoprotein in vascular morphogenesis, the role of granzyme B in modulating angiogenesis is unknown. In the present study, granzyme B cleaved both plasma fibronectin and cell-derived fibronectin, resulting in the release of multiple fibronectin fragments. Granzyme B cleavage of fibronectin resulted in a dose-dependent reduction in endothelial cell adhesion to fibronectin as well as reduced endothelial cell migration and tubular formation. These events were prevented when granzyme B activity was inhibited by a small molecule inhibitor. In summary, granzyme B-mediated cleavage of fibronectin contributes to attenuated angiogenesis through the disruption of endothelial cell — fibronectin interaction resulting in impaired endothelial cell migration and tubular formation.     

A clathrin/dynamin- and mannose-6-phosphate receptor-independent pathway for granzyme B-induced cell death

The 280-kD cation-independent mannose-6-phosphate receptor (MPR) has been shown to play a role in endocytic uptake of granzyme B, since target cells overexpressing MPR have an increased sensitivity to granzyme B-mediated apoptosis. On this basis, it has been proposed that cells lacking MPR are poor targets for cytotoxic lymphocytes that mediate allograft rejection or tumor immune surveillance. In the present study, we report that the uptake of granzyme B into target cells is independent of MPR. We used HeLa cells overexpressing a dominant-negative mutated (K44A) form of dynamin and mouse fibroblasts overexpressing or lacking MPR to show that the MPR/clathrin/dynamin pathway is not required for granzyme B uptake. Consistent with this observation, cells lacking the MPR/clathrin pathway remained sensitive to granzyme B. Exposure of K44A-dynamin-overexpressing and wild-type HeLa cells to granzyme B with sublytic perforin resulted in similar apoptosis in the two cell populations, both in short and long term assays. Granzyme B uptake into MPR-overexpressing L cells was more rapid than into MPR-null L cells, but the receptor-deficient cells took up granzyme B through fluid phase micropinocytosis and remained sensitive to it. Contrary to previous findings, we also demonstrated that mouse tumor allografts that lack MPR expression were rejected as rapidly as tumors that overexpress MPR. Entry of granzyme B into target cells and its intracellular trafficking to induce target cell death in the presence of perforin are therefore not critically dependent on MPR or clathrin/dynamin-dependent endocytosis.     

NK cell protease granzyme M targets alpha-tubulin and disorganizes the microtubule network

Serine protease granzyme M (GrM) is highly expressed in the cytolytic granules of NK cells, which eliminate virus-infected cells and tumor cells. The molecular mechanisms by which GrM induces cell death, however, remain poorly understood. In this study we used a proteomic approach to scan the native proteome of human tumor cells for intracellular substrates of GrM. Among other findings, this approach revealed several components of the cytoskeleton. GrM directly and efficiently cleaved the actin-plasma membrane linker ezrin and the microtubule component alpha-tubulin by using purified proteins, tumor cell lysates, and tumor cells undergoing cell death induced by perforin and GrM. These cleavage events occurred independently of caspases or other cysteine proteases. Kinetically, alpha-tubulin was more efficiently cleaved by GrM as compared with ezrin. Direct alpha-tubulin proteolysis by GrM is complex and occurs at multiple cleavage sites, one of them being Leu at position 269. GrM disturbed tubulin polymerization dynamics in vitro and induced microtubule network disorganization in tumor cells in vivo. We conclude that GrM targets major components of the cytoskeleton that likely contribute to NK cell-induced cell death.     

颗粒酶A诱导Caspases非依赖性细胞死亡

颗粒酶A(granzyme A,GzmA),是存在于细胞毒性T淋巴细胞(CTL)和天然杀伤细胞(NK细胞)的细胞毒颗粒中含量最多的一种丝氨酸蛋白酶,在穿孔素(perforin)协同作用下通过颗粒胞吐(granule exocytosis)释放进入在杀伤细胞和靶细胞之间形成的免疫突触(immunological synapse),然后进入靶细胞的细胞浆,并在细胞核聚集,诱导一种caspases非依赖性细胞死亡。GzmA靶向作用于一种与内质网结合的特殊的复合体——SET复合体,其包含3种GzmA底物：核小体装配蛋白SET、DNA结合蛋白HMG—2、具有碱基切除修复作用的核酸内切酶Apel。SET复合体还含有一种抑癌蛋白pp32和一种具有脱氧核糖核酸酶(DNase)活性的NM23—H1。当GzmA作用于SET复合体时释放出NM23—H1并激活其DNase活性,也阻断了Apel对DNA损伤的修复作用,在DNA上形成单链的缺刻。这是一种新发现的由GzmA诱导的细胞凋亡途径。     

Quantification of the relative importance of CTL, B cell, NK cell, and target cell limitation in the control of primary SIV-infection

CD8+ cytotoxic T lymphocytes (CTLs), natural killer (NK) cells, B cells and target cell limitation have all been suggested to play a role in the control of SIV and HIV-1 infection. However, previous research typically studied each population in isolation leaving the magnitude, relative importance and in vivo relevance of each effect unclear. Here we quantify the relative importance of CTLs, NK cells, B cells and target cell limitation in controlling acute SIV infection in rhesus macaques. Using three different methods, we find that the availability of target cells and CD8+ T cells are important predictors of viral load dynamics. If CTL are assumed to mediate this anti-viral effect via a lytic mechanism then we estimate that CTL killing is responsible for approximately 40% of productively infected cell death, the remaining cell death being attributable to intrinsic, immune (CD8+ T cell, NK cell, B cell) -independent mechanisms. Furthermore, we find that NK cells have little impact on the death rate of infected CD4+ cells and that their net impact is to increase viral load. We hypothesize that NK cells play a detrimental role in SIV infection, possibly by increasing T cell activation.     

The cytotoxic cell protease granzyme B initiates apoptosis in a cell-free system by proteolytic processing and activation of the ICE/CED-3 family protease, CPP32, via a novel two-step mechanism.

The major mechanism of cytotoxic lymphocyte killing involves the directed release of granules containing perforin and a number of proteases onto the target cell membrane. One of these proteases, granzyme B, has an unusual substrate site preference for Asp residues, a property that it shares with members of the emerging interleukin-1beta-converting enzyme (ICE)/CED-3 family of proteases. Here we show that granzyme B is sufficient to reproduce rapidly all of the key features of apoptosis, including the degradation of several protein substrates, when introduced into Jurkat cell-free extracts. Granzyme B-induced apoptosis was neutralized by a tetrapeptide inhibitor of the ICE/CED-3 family protease, CPP32, whereas a similar inhibitor of ICE had no effect. Granzyme B was found to convert CPP32, but not ICE, to its active form by cleaving between the large and small subunits of the CPP32 proenzyme, resulting in removal of the prodomain via an autocatalytic step. The cowpox virus protein CrmA, a known inhibitor of ICE family proteases as well as granzyme B, inhibited granzyme B-mediated CPP32 processing and apoptosis. These data demonstrate that CPP32 activation is a key event during apoptosis initiated by granzyme B.