Serial killing by cytotoxic T lymphocytes: T cell receptor triggers degranulation,re-filling of the lytic granules and secretion of lytic proteins via a non-granule pathway

CD8⁺ cytotoxic T lymphocyte (CTL) clones begin to synthesize the lytic proteins granzyme A, granzyme B and perforin after stimulation with allogeneic target cells. The lytic proteins are stored in the secretory granules which are released after cross-linking of the T cell receptor (TcR) upon target cell recognition. During lytic granule biogenesis granzyme A protein synthesis can be detected between 2 and 10 days after allogeneic stimulation of the CTL. Although granzyme A is stored in the lytic granules over this period, the majority of granzyme A synthesized is secreted directly from the CTL. TcR triggering of degranulation also results in new synthesis of the lytic proteins, which can be inhibited by cycloheximide (CHX). Some of the newly synthesized lytic proteins can be stored in the cell and refill the granules. But up to one third of granzymes A and B can be secreted directly from the CTL via the constitutive secretory pathway as shown by granzyme A enzymatic activity and immunoblots of secreted granzyme B, where one third of the protein fails to acquire the granule targeting signal. Perforin is also secreted via the constitutive pathway, both from the natural killer cell line, YT, and from CTL clones after TcR cross-linking. Constitutive secretion of the lytic proteins can be blocked by both CHX and brefeldin A (BFA). While BFA does not affect the directional killing of recognized targets, it abrogates bystander killing, indicating that bystander killing arises from newly synthesized lytic proteins delivered via a non-granule route. These results demonstrate that the perforin/granzyme-mediated lytic pathway can be maintained while CTL kill multiple targets. We show that CTL not only re-fill their granules during killing, but also secrete lytic proteins via a non-granule-mediated pathway.     

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.     

Characterization of a novel monoclonal antibody against human perforin using transfected cell lines.

The role of perforin in cytotoxicity is controversial. This paper characterizes a novel monoclonal antibody (anti-Phu) against human perforin, using murine cell lines transfected with human perforin cDNA. The antibody specifically stains human perforin in transfected mouse CTLL-2. Anti-Phu blocked granule-mediated haemolysis in an in vitro assay using intact granules isolated from the natural killer (NK)-like human cell line YT, indicating that perforin is a major granule component causing lysis of red blood cells (RBC) in this assay. Inhibition of haemolysis by anti-Phu demonstrated that the antibody binds to undenatured protein as well as fixed perforin molecules. However, the antibody did not inhibit lysis by an allospecific T-cell clone or by YT cells. This could be due to an extremely tight contact between effector and target cell, preventing the antibody from interfering with perforin function by steric hindrance. Physiologically this may reduce lysis of bystander cells. The anti-Phu antibody is a useful tool for further studies of perforin-induced cytotoxicity in vitro and in vivo.     

Protecting a serial killer: pathways for perforin trafficking and self-defence ensure sequential target cell death

Considerable progress has been made in understanding how cytotoxic lymphocytes use the highly toxic pore-forming protein perforin to eliminate dangerous cells, while remaining refractory to lysis. At least two mechanisms jointly preserve the killer cell: the C-terminal residues of perforin dictate its rapid export from the endoplasmic reticulum (ER), whose milieu otherwise favours pore formation; perforin is then stored in secretory granules whose acidity prevent its oligomerisation. Following exocytosis, perforin delivers the proapoptotic protease, granzyme B, into the target cell by disrupting its plasma membrane. Although the precise mechanism of perforin/granzyme synergy remains controversial, the recently defined crystal structure of the perforin monomer and cryo-electron microscopy (EM) of the entire pore suggest that passive transmembrane granzyme diffusion is the dominant proapoptotic mechanism.     

Lymphocyte granule-mediated apoptosis: matters of viral mimicry and deadly proteases

A new form of intercellular signaling is described for lymphocyte granule-mediated apoptosis. Christopher Froelich, Vishva Dixit and Xiaohe Yang propose that perforin delivers granzyme B into target cells by a mechanism analogous to receptor-dependent endocytosis of pathogens. Once in the cytosol, granzyme B prefers to initiate apoptosis by activating the apical protease caspase 10.     

Sulfonyl fluoride serine protease inhibitors inactivate RNK-16 lymphocyte granule proteases and reduce lysis by granule extracts and perforin.

Cytolytic granules purified from natural killer lymphocytes (NK) contain a pore-forming protein (perforin) and a number of serine proteases. When these proteases are inhibited by serine protease-specific isocoumarin reagents the serine proteases are inactivated and the cytolytic activity of the granules is decreased. Paradoxically, it has been found that the general serine protease inhibitor phenylmethylsulfonyl fluoride (PMSF) frequently cannot block killing even though it inhibits many of the serine proteases. At the same time it has been reported that "purified" perforin alone can lyze cells. To address these inconsistencies we first compared the ability of PMSF and four new sulfonyl fluoride serine protease inhibitors to inhibit proteases and cell lysis. We determined the effects on lysis and the second order inhibition rate constants for five granule protease activities: ly-tryptase, ly-chymase, Met-ase (methionine cleaving), Ser-ase (serine cleaving) and Asp-ase (aspartic acid cleaving). One compound, 2-(Z-NH(CH2)2CONH)C6SO2F, was a potent inhibitor of Met-ase activity (k(obsd)/[I] = 162 M-1 s-1), ly-chymase activity (k(obsd)/[I] = 147 M-1 s-1), and granule-mediated as well as perforin-mediated lysis. PMSF was a poor inhibitor of granule proteases (k(obsd)/[I]'s less than 7 M-1 s-1 for four activities and no inhibition of Ser-ase); the lack of reactivity is consistent with the failure of PMSF to block granule lytic activity. We also prepared enriched perforin by anion exchange chromatography and showed that a ly-chymase and a Met-ase associated with perforin. By inhibiting these proteases we also inhibited lytic activity.     

Involvement of granzyme B and perforin gene expression in the lytic potential of human natural killer cells

Natural killer (NK) cells (CD3⁻) or large granular lymphocytes (LGL) spontaneously kill K562 targets but are unable to kill Daudi cells in the absence of IL-2 stimulation. IL-4 is reported to prevent or inhibit the IL-2-driven lymphokine-activated killer (LAK) generation in NK cells. Therefore, we wished to determine whether the antagonistic effect of IL-4 on IL-2-induced LAK activity might regulate the expression of genes encoding proteins involved in lysis, such as perforin, the pore-forming protein, or which are associated with lysis, such as granzymes A and B. By using in situ hybridization, we showed that, in addition to inducing LAK activity, IL-2 stimulation increased the amount of perforin and granzyme B mRNA at the single-cell level in 40 to 100% of the total CD3⁻ LGL cell population. In addition, our results indicated that the stimulatory effect of IL-2 can be downregulated by IL-4 for both LAK activity and granzyme B and perforin gene expression. Here again, a decrease in the amount of specific mRNA per cell was noted. These findings suggest that modulation of the lytic machinery via lymphokines might be associated with regulation of the lytic potential of NK cells.     

Immunohistochemical Expression of Granzyme B and Perforin in Discoid Lupus Erythematosus

Abstract

Discoid lupus erythematosus (DLE) is a chronic photosensitive dermatosis characterized by scarring and atrophy. Granzyme B is a serine protease found in the cytoplasmic granules of cytotoxic lymphocytes and natural killer cells. Perforin permits delivery of the cytotoxic granzymes A and B into target cells to induce apoptosis and cause target cell death. The current study investigated the expression of granzyme B and perforin in 25 cases of DLE and in 10 cases of normal skin by immunohistochemistry and correlated their expression with the clinicopathological features in the studied DLE group. Both granzyme B and perforin were expressed in DLE with absent expression in normal skin. They were parallelly expressed in DLE where granzyme B was associated with features of chronicity such as old age (p?=?0.05) and long duration of the disease (p?=?0.05). Perforin expression in DLE was associated with male gender (p?=?0.04) and outdoor workers (p?=?0.04). Finally, expression of both granzyme B and perforin in dermal lymphocytic inflammatory infiltrate in DLE may indicate the cytotoxicity of the infiltrate. The parallel expression of both molecules may refer to the cooperative relationship between them to enhance cytotoxicity. Higher expression of granzyme B than perforin may indicate the presence of other pathways for granzyme B release independent from perforin.     

Three-color flow cytometric assay for the study of the mechanisms of cell-mediated cytotoxicity

Cytotoxic lymphocytes kill tumor or virus-infected target cells utilizing two mechanisms (1) release of lytic granules (containing perforin and granzymes) and (2) Fas ligand (FasL)/Fas or TNF initiated apoptosis. We have examined mechanisms of target cell lysis using a new Flow Cytometric Cytotoxicity Assay (FC Assay). Target cells were labeled with PKH 67 dye. Cell death was estimated by 7-amino-actinomycin (7-AAD) inclusion and annexin V-PE binding. A strong direct correlation has been found between the percentage of dead target cells in the FC Assay and the results of 51Cr release assay when human LAK and CTL were used as a model system. We have shown that both NK and CTL kill tumor cells mostly by granule-mediated mechanisms, as lysis was blocked by a perforin inhibitor Concanamycin A (Folimycin) but was significantly less sensitive to zVAD-FMK caspase inhibition. The FC assay allows accurate measurement of cell-mediated cytotoxicity as individual target cell death is detected directly.     

Immunology: Expression of perforin, granzyme A and TIA-1 by human uterine CD56+ NK cells implies they are activated and capable of effector functions

At the time the human placenta is established, the uterine mucosallining (decidua) is infiltrated by abundant CD3^– CD56^brightnatural killer (NK) cells. NK cells circulating in blood areknown to contain perforin and granzyme A in their cytoplasmicgranules. TIA-1, an RNA-binding protein capable of inducingDNA fragmentation, has also been found in the granules of cytolyticcells. In this paper, we demonstrate the presence of perforin,granzyme A and TIA-1 in the granules of uterine NK cells. Sixteensections of non-pregnant endometrium throughout the menstualcycle and six sections of early decidua, together with cytospinsof four preparations of isolated decidual leukocytes were stainedby both immuno-histology and immuno-electron microscopy to localizeperforin, granzyme A and TIA-1 to the cytoplasmic granules ofCD56⁺ cells. The presence in vivo of these cytolytic moleculesin a normal physiological situation implies that these uterineNK cells may have effector functions in the control of normalplacentation.     

Purification of natural killer cell cytotoxic granules for assaying target cell apoptosis

We compared two methods originally devised to purify cytoplasmic granules from granulocytes for their capacity to produce cytotoxic granules from natural killer cell lines, suitable for use in target cell apoptosis assays. Both methods utilised nitrogen cavitation to efficiently lyse cells, followed by density gradient fractionation on Percoll to separate the granules from other organelles and granule debris. The first method, originally described by Millard and colleagues, employed DNase I to reduce the viscosity of the initial cell lysate, but the resulting granule fractions were found to contain residual nuclease activity that made them unsuitable for use in apoptosis assays that measure DNA fragmentation. An alternative method described by Borregaard and colleagues utilised a cell relaxation buffer without DNase I. Cytotoxic granules isolated from the NK tumor cell line YT by this protocol were localised predominantly to the densest Percoll fractions, with a density of approximately 1.13 g/ml. These granule fractions were rich in perforin and enzymatically active granzyme B, and induced potent Ca(2+)-dependent lysis and DNA fragmentation of Jurkat cells. Corresponding fractions from non-cytolytic cells, or YT granule extracts incubated with EGTA were unable to mediate significant target cell damage. Cytotoxic granule extracts purified through the Borregaard method were therefore free of nonspecific nuclease activity, and most suitable for studying the mechanism of target cell death induced through the perforin/Ca(2+)-dependent granule pathway.     

Morphological and functional analysis of PTA granules in human NK cells

 Human natural killer (NK) cells contain unique granules with parallel tubular arrays (PTA granules) of approximately 30 nm diameter that can be seen only by electron microscopy. In order to clarify the role of PTA granules in NK cell-mediated cytolysis we examined these structures with regard to frequency and expression of lytic proteins (perforin, granzymes). NK cells (CD3⁻, CD16⁺, CD56⁺) were obtained from heparinized blood of healthy donors and enriched by double-step negative selection using mAb coupled to magnetic beads. PTA granules were found in 31.3% of freshly separated NK cells. When NK cells were cultivated, even in the presence of various stimulating agents (rhIL-2, rhIL-4, rhIL-6, rhIL-12, GM-CSF, rhIFNα, anti-CD16 mAb, dexamethasone), PTA granules disappeared and transformed into conventional primary lysosomes. By immune electron microscopy using antibodies directed against perforin and granzyme B we observed distinct immuno-reactivity in the tubules and in the tubule-associated faintly electron-dense matrix of PTA granules. Immuno-labelling for perforin and granzyme B was also found in the fine granular matrix of primary lysosomes. Finally, we tested the distribution of chondroitin 4-sulfate which is suggested to inactivate lytic proteins. Immuno-reactivity for chondroitin 4-sulfate was detected only in the moderately electron-dense matrix but not in the tubules of PTA granules. These observations indicate that perforin and granzyme B are stored in an inactive form in PTA tubules due to highly ordered paracrystalline protein folding. As soon as the tubules decay the lytic proteins are kept in an environment of chondroitin 4-sulfate for inactivation. Accepted: 24 March 1997     

Nuclear war: the granzyme A-bomb

Granzyme A, a serine protease in the cytotoxic granules of natural killer cells and cytotoxic T lymphocytes, induces caspase-independent cell death when introduced into target cells by perforin. Granzyme A induces single-stranded DNA damage as well as rapid loss of cell membrane integrity and mitochondrial transmembrane potential through unknown mechanisms. Granzyme A destroys the nuclear envelope by targeting lamins and opens up DNA for degradation by targeting histones. A special target of the granzyme A cell death pathway is an endoplasmic reticulum-associated complex, called the SET complex, which contains three granzyme A substrates, the nucleosome assembly protein SET, the DNA bending protein HMG-2, and the base excision repair endonuclease Ape1. The SET complex also contains the tumor suppressor protein pp32 and the granzyme A-activated DNase NM23-H1, which is inhibited by SET. Granzyme A cleavage of SET releases the inhibition and unleashes NM23-H1. Cleavage of Ape1 by granzyme A interferes with the ability of the target cell to repair itself. The novel cell death pathway initiated by granzyme A provides a parallel pathway for apoptosis, important in destroying targets that overexpress bcl-2 or are otherwise invulnerable to the caspases.     

Acquisition of murine NK cell cytotoxicity requires the translation of a pre-existing pool of granzyme B and perforin mRNAs

Although activated murine NK cells can use the granule exocytosis pathway to kill target cells immediately upon recognition, resting murine NK cells are minimally cytotoxic for unknown reasons. Here, we showed that resting NK cells contained abundant granzyme A, but little granzyme B or perforin; in contrast, the mRNAs for all three genes were abundant. Cytokine-induced in vitro activation of NK cells resulted in potent cytotoxicity associated with a dramatic increase in granzyme B and perforin, but only minimal changes in mRNA abundance for these genes. The same pattern of regulation was found in vivo with murine cytomegalovirus infection as a physiologic model of NK cell activation. These data suggest that resting murine NK cells are minimally cytotoxic because of a block in perforin and granzyme B mRNA translation that is released by NK cell activation.     

Suicide induced by cytolytic activity controls the differentiation of memory CD8(+) T lymphocytes

Cytotoxic T lymphocytes (CTL) confer protection against intracellular pathogens, yet the mechanism by which some escape activation induced cell death (AICD) and give rise to long-lived memory cells is unclear. We studied the differentiation of transgenic TCR CD8(+) cells into CTL and memory cells using a novel system that allowed us to control cytolytic activity. The perforin/granzyme granules used to lyse targets induced the apoptosis of CTL in a fratricide-independent manner. After adoptive transfer to antigen-free mice, the ability of CTL to give generate memory cells was determined. We found that the extent of cytolysis by a common pool of CTL controlled the differentiation into memory cells, which were only generated under conditions of minimal cytolytic activity. Thus, the differentiation of naive CD8(+) cells into memory cells may not depend on the presence on a subset of committed CTL precursors, but rather is controlled by the extent of granule-mediated cytolysis.     

Involvement of granzyme B and perforin gene expression in the lytic potential of human natural killer cells

Natural killer (NK) cells (CD3-) or large granular lymphocytes (LGL) spontaneously kill K562 targets but are unable to kill Daudi cells in the absence of IL-2 stimulation. IL-4 is reported to prevent or inhibit the IL-2-driven lymphokine-activated killer (LAK) generation in NK cells. Therefore, we wished to determine whether the antagonistic effect of IL-4 on IL-2-induced LAK activity might regulate the expression of genes encoding proteins involved in lysis, such as perforin, the pore-forming protein, or which are associated with lysis, such as granzymes A and B. By using in situ hybridization, we showed that, in addition to inducing LAK activity, IL-2 stimulation increased the amount of perforin and granzyme B mRNA at the single-cell level in 40 to 100% of the total CD3- LGL cell population. In addition, our results indicated that the stimulatory effect of IL-2 can be downregulated by IL-4 for both LAK activity and granzyme B and perforin gene expression. Here again, a decrease in the amount of specific mRNA per cell was noted. These findings suggest that modulation of the lytic machinery via lymphokines might be associated with regulation of the lytic potential of NK cells.     

Expression of granzyme B in peripheral blood polymorphonuclear neutrophils (PMN), myeloid cell lines and in PMN derived from haemotopoietic stem cells in vitro

Granzyme B and perforin are the major protagonists of cytotoxicity mediated by natural killer (NK) cells or cytotoxic T cells. More recent we described the presence of granzyme B and perforin in polymorphonuclear neutrophils (PMN), a finding in discrepancy with the credo that granzyme B and perforin expression is restricted to cytotoxic T cells and NK cells. In extension of our previous study, we now provide evidence that granzyme B is not only present in mature PMN, but also in the myeloid cell lines HL-60 and U937, in CD34+ stem cells, and in PMN derived from CD34+ cells in vitro. In agreement with the "targeting by time" hypothesis we found the bulk of granzyme B in association with primary granules, in addition to a minor membrane expression. Granzyme B, on one hand might, enhance the cytotoxic potential of PMN, on the other, it may provide PMN with additional means to degrade extracellular matrices.     

Cytotoxic cell granule-mediated apoptosis: perforin delivers granzyme B-serglycin complexes into target cells without plasma membrane pore formation

The mechanism underlying perforin (PFN)-dependent delivery of apoptotic granzymes during cytotoxic cell granule-mediated death remains speculative. Granzyme B (GrB) and perforin were found to coexist as multimeric complexes with the proteoglycan serglycin (SG) in cytotoxic granules, and cytotoxic cells were observed to secrete exclusively macromolecular GrB-SG. Contrary to the view that PFN acts as a gateway for granzymes through the plasma membrane, monomeric PFN and, strikingly, PFN-SG complexes were shown to mediate cytosolic delivery of macromolecular GrB-SG without producing detectable plasma membrane pores. These results indicate that granule-mediated apoptosis represents a phenomenon whereby the target cell perceives granule contents as a multimeric complex consisting of SG, PFN, and granzymes, which are, respectively, the scaffold, translocator, and targeting/informational components of this modular delivery system.