Subcellular localization of perforin and serine esterase in lymphokine-activated killer cells and cytotoxic T cells by immunogold labeling

CTL, NK cells, and lymphokine-activated killer (LAK) cells are cytolytic lymphocytes known to produce a pore-forming protein, named perforin or cytolysin, that lyses target cells by forming large pores on the plasma membrane of the target cell. Other proteins besides perforin are found in the cytoplasmic granules of effector lymphocytes, and these include a family of serine esterases. Ultrastructural immunogold labeling studies with antibodies against perforin and a serine esterase (MTSP-1, also known as granzyme A and SE-1) show that all the granules of LAK cells and a CTL cell line contain perforin and serine esterase. For both LAK cells and CTL, perforin has been located mostly in the fine granular matrix of the granules, whereas gold particles corresponding to serine esterase have been found in both the matrix and the cap regions of the granules. Results from double immunogold labeling indicate that perforin and serine esterase colocalize to the same granules.     

Immunogold labeling of perforin and serine esterases in granulated metrial gland cells

Cytotoxic T lymphocytes (CTL) and natural killer (NK) cells are cytolytic lymphocytes known to produce a pore-forming protein, named perforin or cytolysin, that lyses target cells by creating large pores on the target plasma membrane. Besides perforin, the granules of CTL and NK cells contain a family of serine esterases. Perforin has also been localized in granulated metrial gland (GMG) cells of the murine embryo implantation site by light microscopic immunostaining. Ultrastructural immunogold labeling with antibodies against perforin and a serine esterase (MTSP 1 or granzyme A) shows that GMG cells contain both perforin and serine esterases in the fine granular matrix of their granules. Perforin has been located in all of the granules, whereas gold particles corresponding to serine esterases have been found in most of the granules. Results from the double immunogold technique indicate that perforin and serine esterases colocalize to most of the same granules in GMG cells. This study supports the view that GMG cells are related to cytolytic lymphocytes.     

Characterization of granzymes A and B isolated from granules of cloned human cytotoxic T lymphocytes

A human CD8+ CTL clone with cytolytic potential was shown to express two serine proteases, a 50-kDa homodimer and a 27-kDa monomer, which were purified from cytoplasmic granules. N-terminal sequencing of the purified proteins revealed that the 50-kDa homodimer is the gene product of the human Hanukah factor cDNA clone and that it represents the human homologue to granzyme A. Similarly, the 27-kDa protein was shown to be the serine esterase encoded by the human lymphocyte protease cDNA clone and corresponds to granzyme B. There was no evidence for the presence of other granzymes, in particular for the human homologues to murine granzymes C, D, E, and F. The substrate best cleaved by granzyme A was Gly-Pro-Arg-amido-4-methyl-coumarin after the Arg residue and the pH optimum was 8 to 8.5. Upon triggering of the TCR-CD3 complex with an anti-CD3 mAb, granzyme A was released into the culture medium. Furthermore, a granule-associated hemolytic activity was detected after salt extraction and partial purification of granule proteins. This suggests that hemolytically active human perforin can be obtained from inactive granules.     

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。     

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.     

Induction of perforin and serine esterases in a murine cytotoxic T lymphocyte clone

The expression of perforin and serine esterase (SE) activities and genes was examined in a murine cytotoxic T lymphocyte line (R8i) that does not require exogenous IL-2 for proliferation. Although perforin (hemolytic) activity was detected in unstimulated R8i, it was induced 2- to 14-fold in the presence of IL-2, IL-3, IL-4, and IL-6, and to a lesser degree (less than 4-fold) by TNF and IFN-gamma. A transient induction was also observed at the mRNA level. Peak perforin protein and mRNA levels were reached within 24 h and started to decline 48 h after stimulation. A trypsinlike SE activity which cleaves the chromogenic substrate N, alpha-benzyloxycarbonyl-L-lysine thiobenzyl ester was also induced 2- to 4-fold in the presence of the various IL tested. At the mRNA level, the message for SE SE1/granzyme A/Hanukah factor was absent from R8i whereas SE2/granzyme B/CTLA-1 increased by greater than 3-fold in the presence of IL-2, IL-3, IL-4, and IL-6 and occurred with the same kinetics and pattern as perforin. The induction response occurred without any enhancement of cell proliferation, suggesting that the cytokines tested may provide a direct differentiation signal to CTL. The induction response was abrogated effectively by inhibitors of protein (cycloheximide or emetine) and RNA (actinomycin D) syntheses. These findings suggest that the various IL may provide both a growth signal and a differentiation signal to CTL, resulting in the direct activation of perforin and SE genes.     

Purification of a membrane-associated serine esterase from murine cytotoxic T lymphocytes by a single reverse-phase column

The plasma and organelle membranes of a cytotoxic T lymphocyte line, CTLL-R8, were isolated by subcellular fractionation. After dissolving in detergent-containing buffer, the membrane proteins were isolated by high-performance liquid chromatography on a single reverse-phase column. The serine esterase activity in the fractions was detected by measuring hydrolysis of the ester compound N alpha-benzyloxycarbonyl-L-lysine thiobenzyl ester. A major band was revealed in the fraction with highest serine esterase activity. Under sodium dodecyl sulfate-polyacrylamide gel electrophoresis, this band assumes a molecular weight of about 30 kDa. The amino-terminal sequence of the protein was analyzed and shows 100% identity with that of MCSP-3/granzyme F, a soluble serine esterase previously identified in the cytoplasmic granules of cytotoxic T lymphocytes. Modifications of this reverse-phase column method would thus represent a simple, convenient strategy for obtaining high yields of all the lymphocyte surface proteases, which could then be further characterized for function.     

Granulysin delivered by cytotoxic cells damages endoplasmic reticulum and activates caspase-7 in target cells

Granulysin is a human cytolytic molecule present in cytotoxic granules with perforin and granzymes. Recombinant 9-kDa granulysin kills a variety of microbes, including bacteria, yeast, fungi, and parasites, and induces apoptosis in tumor cells by causing intracellular calcium overload, mitochondrial damage, and activation of downstream caspases. Reasoning that granulysin delivered by cytotoxic cells may work in concert with other molecules, we crossed granulysin transgenic (GNLY(+/-)) mice onto perforin (perf)- or granzyme B (gzmb)-deficient mice to examine granulysin-mediated killing in a more physiologic whole-cell system. Splenocytes from these animals were activated in vitro with IL-15 to generate cytolytic T cells and NK cells. Cytotoxic cells expressing granulysin require perforin, but not granzyme B, to cause apoptosis of targets. Whereas granzyme B induces mitochondrial damage and activates caspases-3 and -9 in targets, cytotoxic cell-delivered granulysin induces endoplasmic reticulum stress and activates caspase-7 with no effect on mitochondria or caspases-3 and -9. In addition, recombinant granulysin and cell-delivered granulysin activate distinct apoptotic pathways in target cells. These findings suggest that cytotoxic cells have evolved multiple nonredundant cell death pathways, enabling host defense to counteract escape mechanisms employed by pathogens or tumor cells.     

Heterogeneity of long-term cultured activated killer cells induced by anti-T3 antibody

The present study has characterized the short term and long term cultured murine-activated killer (AK) cells that are induced by antibody directed against the epsilon-chain of T3 complex. The conventional lymphokine AK (LAK) cells were generated by culturing normal B6 spleen cells with purified human rIL-2. The alpha T3-induced AK cells (T3AK) were induced by culturing normal B6 spleen cells with alpha T3 and were then maintained in culture medium supplemented with human rIL-2 and/or alpha T3. After initial activation with alpha T3, lymphocyte proliferation and generation of cytotoxic effectors (T3AK) were noted, and these events were related to the endogenous production of IL-2 and IL-4. Addition of alpha IL-2 and/or alpha IL-4 suppressed both the proliferative response and the cytotoxic response induced by alpha T3. In comparing the T3AK cells with the conventional LAK cells, there were many similarities as well as some distinct differences. Both cells displayed a similar cytotoxic spectrum against a variety of tumor targets. The T3AK cells usually gave much higher levels of cytotoxic activity against susceptible targets. However, the susceptibility of different tumor targets to conventional LAK cells and T3AK cells varied. The time course for the generation of lytic activity also differed between the conventional LAK and T3AK cells. One distinct difference was their ability to survive in vitro. The conventional LAK cells survived in culture for only 1 wk. The T3AK cells could survive for at least 4 to 5 wk with active growth. The serologic phenotype of the LAK precursors was asialo GM1 (AsGM1+) cells, but the T3AK precursors could be either AsGM1+ or AsGM1-, depending on the target cell. The LAK effectors were both Lyt-2+ and Lyt-2-, but the short-term T3AK effectors were exclusively Lyt-2+. The long term T3AK cells (cultured for more than 2 wk) were found to consist of Lyt-2+ and Lyt-2- cells, and these subsets of T3AK cells showed different target specificities. These findings demonstrate the heterogeneity of LAK and T3AK cells, and this heterogeneous property may contribute to their diversity in specificity against different tumor targets and thus may affect their effectiveness in the immunotherapy of cancer.     

Design Parameters for Granzyme-Mediated Cytotoxic Lymphocyte Target-Cell Killing and Specificity

Cytotoxic lymphocytes are key elements of the immune system that are primarily responsible for targeting cells infected with intracellular pathogens, or cells that have become malignantly transformed. Target cells are killed mainly via lymphocyte exocytosis of specialized lysosomes containing perforin, a pore-forming protein, and granzymes, which are proteases that induce apoptosis. Due to its central role in lymphocyte biology, as well as its implication in a host of pathologies from cancer to autoimmunity, the granzyme-perforin pathway has been the subject of extensive investigation. Nevertheless, the details of exactly how granzyme and perforin cooperate to induce target-cell death remain controversial. To further investigate this system, we developed a biophysical model of the immunological synapse between a cytotoxic lymphocyte and a target cell using a spatial stochastic simulation algorithm. We used this model to calculate the spatiotemporal evolution of granzyme B and perforin from the time of their exocytosis to granzyme internalization by the target cell. We used a metric of granzyme internalization to delineate which biological processes were critical for successful target-cell lysis. We found that the high aspect ratio of the immunological synapse was insufficient in this regard, and that molecular crowding within the synapse is critical to preserve sufficient concentrations of perforin and granzyme for consistent pore formation and granzyme transfer to target cells. However, even when pore formation occurs in our model, a large amount of both granzyme and perforin still escape from the synapse. We argue that a tight seal between the cytotoxic lymphocyte and its target cell is not required to avoid bystander killing. Instead, we propose that the requirement for spatiotemporal colocalization of granzyme and perforin acts as an effective bimolecular filter to ensure target specificity.     

Cytotoxicity with target DNA breakdown by rat basophilic leukemia cells expressing both cytolysin and granzyme A.

The noncytotoxic rat mast cell tumor line RBL was transfected with genes for the cytotoxic lymphocyte granule proteins cytolysin (perforin) and granzyme A, giving transfectants with mRNA and protein expression levels comparable with cloned cytotoxic T lymphocytes. Both RBL-cytolysin and RBL-cytolysin-granzyme A transfectants showed extremely potent killing of red cell targets and lysed 20%-60% of EL4 lymphoma targets at an effector-to-target ratio of 30. RBL transfectants expressing only granzyme A were not cytotoxic. Significant EL4 DNA breakdown accompanying lysis was observed only with RBL that was transfected with both cytolysin and granzyme A. These results support the granule-exocytosis model for lymphocyte cytotoxicity and show that effector granzyme A plays a role in target cell DNA breakdown.     

Regulation of lymphokine-activated killer activity and pore-forming protein gene expression in human peripheral blood CD8+ T lymphocytes. Inhibition by transforming growth factor-beta.

The effect of transforming growth factor-beta 1 (TGF-beta) on activation-induced CD8+ T cell cytotoxicity and gene expression was investigated. TGF-beta was demonstrated to inhibit pore-forming protein (PFP) mRNA expression and total benzoyloxycarbonyl-L-lysine thiobenzyl ester esterase activity in CD8+ T cells cultured with IL-2 and OKT3 mAb for 6 to 18 days. Consistently, in the absence or presence of TGF-beta, the PFP mRNA expression and lymphokine-activated killer (LAK) activity of CD8+ T cells were closely correlated. The inhibitory effects of TGF-beta on both CD8+ T cell PFP mRNA expression and LAK activity were reversible by removal of TGF-beta from the culture. Expression of lymphokines, adhesion/recognition molecules, and activated p55 IL-2R, previously implicated in the lytic mechanism of cytotoxic lymphocytes, either was not detectable or did not correlate with TGF-beta inhibition of LAK activity. In addition, independently of effector/target cell binding, the lectin- or heteroconjugated antibody-dependent cellular cytotoxicity of IL-2/OKT3 mAb-activated CD8+ T cells was inhibited by preculture with TGF-beta. TGF-beta also inhibited the rapid activation-induced expression of PFP mRNA and cytotoxic potential in resting T cells, thereby indicating that the effect of TGF-beta was independent of T cell proliferation. TGF-beta inhibition of CD8+ T cell PFP mRNA expression and cytotoxic potential was TGF-beta dose dependent; however, a variety of activation stimuli (including IL-2, IL-6, and OKT3 mAb) were all similarly inhibited by TGF-beta. Therefore, TGF-beta may be an important general regulator of CD8+ T cell cytotoxic function, in particular by suppressing expression of PFP, a major cytolytic protein implicated in the lytic function of cytotoxic lymphocytes.     

Antiviral effect of lymphokine-activated killer cells: characterization of effector cells mediating prophylaxis

Lymphokine-activated killer (LAK) cells generated by cultivation of C57BL/6 mouse spleen cells in the presence of recombinant interleukin-2 were transferred into natural killer (NK) cell-deficient suckling mouse recipients. These mice were then challenged with either murine cytomegalovirus (MCMV) or lymphocytic choriomeningitis (LCMV) and sacrificed 3 days later. No interleukin 2 infusions were given. Mice receiving as few as 5 x 10(5) LAK cells had several 100-fold decreases in spleen MCMV titers as compared with untreated mice. This treatment had no effect on spleen LCMV titers. The LAK cell cultures contained 10 to 17% NK 1.1+, 50 to 55% Lyt-2+, and 33 to 50% immunoglobulin D+ cells. Double fluorescence labeling and in vitro cytotoxicity assays with fluorescence-activated cell sorting revealed at least two mutually exclusive killer cell populations. NK 1.1+ LAK cells resembled freshly isolated activated NK cells with regard to target cell range (YAC-1 cell killing greater than L-929, P815, and EL-4 cell killing), large granular lymphocyte (LGL) morphology, and decreased ability to lyse interferon (IFN)-treated target cells. Lyt-2+ LAK cells lysed the targets mentioned above but at lower levels and without the differences in susceptibility mentioned above. These Lyt-2+ LAK cells also had a decreased ability to lyse IFN-treated targets, in contrast to classic cytotoxic T lymphocytes, which lyse IFN-treated targets far more efficiently than untreated targets. Purified populations of LAK cells obtained by fluorescence-activated cell sorting were used in the antiviral protection model. The results showed that protection against MCMV could be mediated by NK 1.1+, NK 1.1-, Lyt-2+, Lyt-2-, and IgD- populations but not by IgD+ cells. The five protective populations all had in common the LGL phenotype and cytotoxic activity in vitro. The IgD+ population did not contain LGLs, lyse target cells in vitro, or mediate an antiviral effect in vivo. These results suggest that LAK cells may be therapeutically useful against certain virus infections (MCMV) but not others (LCMV) and that despite their heterogeneity in antigenic phenotype and cytotoxic activity, their pattern of antiviral activity in vivo resembles that of NK cells, which protect against MCMV but not LCMV.     

Recognition and lysis of target cells by cytotoxic T lymphocytes

A single cytotoxic T lymphocyte (CTL) is capable of performing the two most fundamental functions of an immune response, recognition and elimination of foreign antigens. It is now clear that in a CTL these two functions are linked via the antigen-specific, heterodimeric receptor. We review here some experimental approaches that justify this conclusion and provide the means for further examination of the mechanisms by which CTLs lyse their target cells. When antireceptor antibodies serving as antigen substitutes are attached to various cells, they trigger the lytic activity of particular CTLs, which results in lysis of the antibody-modified cell. In the process, a novel serine esterase, which is located within cytolytic granules of the CTL, is released. The presence of this enzyme and a complement-like protein, perforin, in granules of a CTL has led to the suggestion that CTLs and complement have similar cytolytic mechanisms. However, the resistance of some CTLs to lysis by other CTLs, but not to lysis by antibody-activated complement, suggests fundamental differences between cytolytic mechanisms of CTLs and complement.     

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.     

Granzyme A binding to target cell proteins. Granzyme A binds to and cleaves nucleolin in vitro.

The physiologic substrates of cytotoxic T lymphocyte granule-associated serine esterases (referred to hereafter as proteases or "granzymes"), and the role of these enzymes in cell-mediated activity remain unclear. We have developed an assay for possible ligands of the trypsin-like dimeric serine protease granzyme A based on Western immunoblotting techniques. This protein-binding assay demonstrates the selective binding of granzyme A to several proteins present in the target cell P815. The binding specificity is preserved when enzyme binding is performed in the presence of excess competing proteins, including such cationic species as lysozyme and RNase. Enzyme binding is inhibited, however, by heat or detergent inactivation of granzyme A. Subcellular fractionation of target cells shows that the nuclear fraction contains most granzyme A binding reactivity, which is recovered in the nuclear salt wash fraction. A protein with Mr = 100,000 and two closely migrating proteins with Mr = 35,000 and 38,000 are the predominant reactive moieties, and the N-terminal sequence of the 100-kDa protein confirmed that this protein was murine nucleolin. Incubation of granzyme A with nucleolin generates a discrete proteolytic cleavage product of Mr = 88,000. Since nucleolin is known to shuttle between nucleus and cytoplasm, the interaction of granzyme A and nucleolin may be important in the process of apoptosis which accompanies cytotoxic T lymphocyte-mediated lysis of target cells.     

Expression of mRNAs for pore-forming protein and two serine esterases in murine primary and cloned effector lymphocytes

The cDNAs encoding several proteins present in the granules of cytolytic effector lymphocytes have now been cloned. These include the cytolytic pore-forming protein (PFP) or perforin, and at least six serine esterases (SE), also called granzymes. The cDNA probes for PFP, SE-1, and SE-2 are used here to study the expression of these proteins in murine primary effector lymphocytes. Among the stimuli effective in inducing the expression of PFP, SE-1, and SE-2 were recombinant interleukin-2, the lectin concanavalin A in the presence of phorbol esters, and allogeneic cells in mixed lymphocyte cultures. Some correlation was seen between the levels of PFP and SE mRNAs and cytotoxicity measured in a standard 51Cr release assay. We also examined a panel of 13 cloned cytotoxic T lymphocyte (CTL) lines and found that mRNAs for PFP and SE-2 were expressed in all CTL lines, including some that were previously considered not to produce PFP. Twelve of the 13 CTL lines also proved to possess the mRNA for SE-1. One thymoma cell line, TIMI.4, did not express mRNA for PFP, although it expressed mRNA for SE-1 and SE-2.     

Induction of rapid histone degradation by the cytotoxic T lymphocyte protease Granzyme A

The cytotoxic T lymphocyte protease granzyme A induces caspase-independent cell death in which DNA single-strand nicking is observed instead of oligonucleosomal fragmentation. Granzyme A is a specific tryptase that concentrates in the nucleus of targeted cells and synergistically enhances DNA fragmentation induced by the caspase activator granzyme B. Here we show that granzyme A treatment of isolated nuclei enhances DNA accessibility to exogenous endonucleases. In vitro and after cell loading with perforin, GrnA completely degrades histone H1 and cleaves core histones into approximately 16-kDa fragments. Histone digestion provides a mechanism for unfolding compacted chromatin and facilitating endogenous DNase access to DNA during T cell and natural killer cell granule-mediated apoptosis.     

Modulation of perforin, granzyme A, and granzyme B in murine natural killer (NK), IL2 stimulated NK, and lymphokine-activated killer cells by alcohol consumption.

Alcohol consumption in mice suppresses the cytolytic activity of natural killer (NK) and lymphokine-activated killer (LAK) cells through unknown mechanisms. Herein, we found that alcohol consumption decreased target cell-induced release of granzyme A activity in freshly isolated splenic NK cells, in NK cells stimulated for 18 h with 1000 IU/ml of interleukin 2, and in LAK cells. The total activity and protein expression of granzymes A and B also were lower in these cells than in cells isolated from water-drinking mice. Interleukin 2 increased granzyme A protein expression independent of alcohol consumption; however, this increase was associated with decreased enzyme activity. In contrast, granzyme B protein expression and enzymatic activity increased in response to interleukin 2. Perforin activity and protein expression were reduced in LAK cells generated from alcohol-consuming mice. We conclude that the mechanism underlying the suppression of NK and LAK cytolytic activity by alcohol consumption involves the collective reduction of target-induced release, activity, and expression of perforin and granular proteases.