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*
Departamento de Bioquimica y Biologia Molecular y Cellular, Facultad de Ciencias, Universidad de Zaragoza, Zaragoza, Spain; and
Division of Immunology and Transplantation Biology, Department of Pediatrics, Stanford University School of Medicine, Stanford, CA 94305
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cytolysis induced by both granule-mediated and Fas-based mechanisms exhibits the typical features of apoptosis (9). The biochemical mechanism for Fas-induced apoptosis involves the recruitment and activation of intracellular cysteine proteases of the caspase family (10). In addition, granzyme B can cleave and activate several caspases (11, 12). Although nuclear manifestations of granzyme B-induced apoptosis are dependent on caspase activation (7, 13, 14, 15), cytolysis appears to be caspase independent (13, 15). In contrast, the manifestations of granzyme A-induced apoptosis appear independent of caspase activation (14).
Granulysin is a protein localized inside CTL granules with an expression pattern similar to those of perforin and granzymes, being selectively up-regulated during the stage at which these cells are able to exert cytolytic function (16, 17). This protein is exocytosed after CTL stimulation through the TCR and exerts lytic activity as measured by 51Cr release assays (17). These data suggest that granulysin-induced cytolysis could constitute another granule-associated lytic pathway in addition to the perforin/granzyme pathway (17). Granulysin is highly homologous to another lytic molecule localized inside porcine NK cell granules, termed NK-lysin (18), and to Entamoeba histolytica amoebapores (19), proteins capable of forming pores into lipid membranes. These homologies suggest that granulysin might also form pores. Granulysin also shares sequence homology with saposins (17). Saposins are not pore-forming proteins, but are proteins that interact with lipid membranes and activate lipid-degrading enzymes (specifically, glucosylceramidases and sphingomyelinases) (20, 21). The immediate consequence of activation of these enzymes is the increase in cellular ceramide content (22). Ceramide, of note, has been proposed as a mediator in some apoptotic processes (23, 24), and when added exogenously, ceramide can induce apoptosis (25, 26).
The partial homology of granulysin with saposins suggested that it might induce cytotoxicity through activation of sphingomyelinases and an increase in cellular ceramide content. Here we demonstrate that granulysin causes apoptotic cell death. Jurkat tumor cells treated with recombinant granulysin exhibit a significant increase in ceramide content and a decrease in sphingomyelin. However, granulysin also caused apoptosis in cells depleted of sphingomyelin by prolonged treatment with the ceramide synthase inhibitor fumonisin B1. This finding suggests that granulysin may induce apoptosis via more than one pathway.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The Jurkat T cell leukemia (American Type Culture Collection, Rockville, MD; clone E6.1) was used as the target in the granulysin- and ceramide-induced cytotoxicity assays performed in this study. Jurkat cells were cultured in RPMI 1640 medium (BioWhittaker, Barcelona, Spain) supplemented with 5% FCS, L-glutamine, and penicillin/streptomycin (hereafter, complete medium) using standard cell culture procedures.
Materials
Fumonisin B1 from Fusarium moniliforme, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT),3 ceramide type III from bovine brain, and dodecane were purchased from Sigma (Madrid, Spain). N-acetyl-Asp-Glu-Val-Asp aldehyde (Ac-DEVD-CHO) and benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD-fmk) were obtained from Bachem (Bubendorf, Switzerland). [1-14C]palmitic acid was purchased from Amersham (Madrid, Spain). Mouse IgG2a anti-human CPP32 (caspase 3, clone 19) was obtained from Transduction Laboratories (Affiniti, Mamhead, U.K.).
Expression and purification of recombinant functional granulysin
Recombinant granulysin was expressed and purified essentially as previously described (17). Briefly, a portion of the 519 cDNA coding for glycine residue 63 through the arginine residue 136 of P520 (27) followed by a stop codon was cloned into the pET28a vector (Novagen, Madison, WI) to express polyhistidine-tagged recombinant 9-kDa granulysin in Escherichia coli BL21 (DE3). A control construct of similar size, coding for residues 549 through 646 of human heat shock protein 70 (HSC70) was produced for parallel expression and purification. The recombinant proteins were expressed and purified on a nickel column under denaturing conditions according to Novagen’s instructions. After purification, the proteins were reduced by the addition of 1 mM DTT, allowed to refold in the presence of oxidized DTT, and dialyzed against Tris-buffered saline; the histidine tag removed by thrombin treatment and purified by reverse phase HPLC. Quantification of the protein was performed using a standard commercial kit from Bio-Rad (Richmond, CA) with lysozyme as a standard. Protein purity was always >95%, as assessed by Coomassie staining of 15% SDS-PAGE gels.
Cytotoxicity assays
Jurkat cells were seeded in flat-bottom, 96-well plates at an
initial density of 2 x 105 cells/ml (100 µl/well)
and cultured for 16 h in complete medium in the presence or the
absence of 25 ng/ml of the cytotoxic anti-Fas mAb CH-11, as
described previously (26, 28). For granulysin-induced lysis, cells were
similarly resuspended in RPMI 1640 medium supplemented with 1% FCS and
incubated for 16 h at 37°C in the presence or the absence of 20
to 50 µM recombinant granulysin. Natural (C18) ceramide
(50–200 nM) was first dissolved in ethanol/dodecane (98/2, v/v) and
then added to serum-free culture medium (RPMI 1640/DMEM/Ham’s F-12,
2/1/1) as previously described (26, 29). At the doses used, ethanol and
dodecane had no effect on cell proliferation. Ac-DEVD-CHO, an inhibitor
of CPP32-like caspases (30), or Z-VAD-fmk, a more general caspase
inhibitor (31), were used at 600 and 100 µM, respectively. Peptide
inhibitors were first dissolved in DMSO and diluted in culture medium
(final DMSO concentration, 
0.4%, v/v). The addition of the peptide
inhibitors at concentrations up to 1.2 mM did not affect to the growth
rate or cell morphology of Jurkat cells. Cells were preincubated for
1 h with the protease inhibitors before adding the toxic stimuli
to assure a sufficient incorporation by cells (26, 28, 32). Jurkat
cells were depleted of sphingomyelin and ceramide by culture for 3 days
in complete medium supplemented with 50 µM fumonisin B1,
a potent inhibitor of ceramide synthase (33). Cell viability was
determined by a modification of the MTT reduction method of Mosmann
(34) and expressed as a percentage of that in control cultures. Cell
death was determined by the trypan blue exclusion test and microscopy
of stained cells. Phosphatidylserine (PS) exposure during apoptosis was
evaluated by annexin V-FITC staining (35). Briefly, cells were washed
with PBS and incubated in a solution of 0.5 µg/ml FITC-labeled
annexin V (Bender, Barcelona, Spain) in binding buffer (140 mM NaCl,
2.5 mM CaCl2, and 10 mM HEPES/NaOH, pH 7.4) at 4°C for 30
min. Cells were then centrifuged, washed, resuspended in 1 ml of
binding buffer, and analyzed by flow cytometry.
Ceramide and sphingomyelin determinations
Ceramide and sphingomyelin levels were assayed essentially as previously described (26). Jurkat cells (1.8 x 106 in 10 ml) were labeled for 48 h with 5 µCi of [1-14C]palmitic acid bound to fatty acid-free serum albumin (1/1, molar ratio) in complete medium. Cells were resuspended in RPMI 1640 medium supplemented with 1% FCS at 4 x 105 cells/ml, and granulysin (50 µM) or the control recombinant protein HSC70 was added. After incubation for 16 h at 37°C, the cells were harvested, washed with cold RPMI medium, and counted. Cell viability was determined in an aliquot of the corresponding cell suspensions by the MTT assay. Total cell lipids were extracted at 4°C with chloroform/methanol (2/1, v/v) (36). Radioactivity in aliquots from chloroformic phases was determined by liquid scintillation counting, and equal amounts of radioactivity for each sample were applied to TLC Silica gel G plates (Scharlau, Barcelona, Spain). Plates were prewashed with chloroform/methanol (1/1, v/v) and heat-activated at 110°C for 1 h. A first development of TLC-loaded samples was performed with chloroform/methanol/water (60/30/5, v/v) up to 10 cm from the bottom of the plate, and a second development was performed to its full length with hexane/diethyl ether/acetic acid (80/20/2, v/v/v). Plates were air-dried, and radiolabeled bands were located by film autoradiography (Hyperfilm ß-max, Amersham) at room temperature for 2 to 3 days. Then, sample lanes were covered with glass, allowing the marker lanes to be iodine stained. Standards of ceramide, sphingomyelin, cholesterol, phosphatidylcholine, and PS (Sigma) were used as markers. The corresponding radiolabeled lipids were scraped and transferred to vials, to which 4 ml of scintillation mixture (Normascint 11, Scharlau) containing 10% (v/v) methanol were added. Radioactivity in samples was determined by liquid scintillation counting, and results were expressed as ceramide/sphingomyelin (Cer/SM) ratios.
Fluorescence microscopy
Morphologic evaluation of apoptosis was monitored by cell labeling with the nuclear stain p-phenylenediamine (PPDA) and then visualized by fluorescence microscopy (37). Briefly, cells were washed with PBS, pH 7.4, and fixed at room temperature with 1% paraformaldehyde in PBS for 15 min. Fixed cells were washed with PBS, centrifuged onto glass coverslips in wells of a 24-well plate, mounted on a glass slide over a drop of PPDA stain (10 mg of PPDA in 1 ml of PBS mixed with 9 ml of oxidized glycerol) (28), and photographed.
Analysis of CPP32 activation by immunoblotting
CPP32 activation was evaluated by Western blot analysis of cell homogenates with a specific anti-human CPP32 Ab (38). Jurkat cells (5 x 106 in 1 ml) were treated with 100 nM ceramide, as indicated above, for 5 h at 37°C. At the end of the incubations, cells were recovered by centrifugation at 4°C, washed twice with cold PBS, and lysed in 1 ml of lysis buffer (50 mM Tris/HCl, pH 7.6, containing 1% Triton X-100, 0.15 M NaCl, 1 mM EDTA, 30 mM NaF, 10 µg/ml leupeptin, and 1 mM PMSF), as previously described (39). Cell lysates were centrifuged (4°C, 12,000 x g, 15 min), and solubilized proteins were separated by SDS-PAGE in an SDS-12%-polyacrylamide gel under reducing conditions and transferred to nitrocellulose membranes (Hybond-C Extra, Amersham). Membranes were then sequentially incubated with 50 ng/ml anti-CPP32 Ab in PBS containing 5% BSA and with 0.2 µg/ml goat anti-mouse IgG coupled to alkaline phosphatase (Sigma) for 1 h and revealed with 5-bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazolium (40).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recombinant granulysin is cytotoxic by itself in a
51Cr release assay using B lymphoblastoid and other target
cells (17). If this lysis were due to pore formation in the plasma
membrane and disruption of osmotic equilibrium, as has been described
for NK-lysin and amoebapores (18, 19), necrotic, rather than apoptotic,
cell death should be observed. Jurkat cells were treated with various
doses of recombinant granulysin, and toxicity was tested by the MTT
assay. The IC50 dose was 40 µM, with no toxicity at 10
µM and almost 100% cell death at 100 µM. This dose response is
similar to that previously obtained for granulysin-induced
51Cr release from YAC cells (17). To test the type of cell
death induced by granulysin, Jurkat cells were treated with 50 µM
recombinant granulysin or anti-Fas mAb, and nuclei were stained
with the fluorescent probe PPDA. Both granulysin and
anti-Fas mAb induced nuclear features of typical apoptosis: cell
shrinkage, blebbing, chromatin condensation, and nuclear fragmentation
(Fig. 1
). Another early event that takes
place during apoptosis is the redistribution of PS from the inner to
the outer leaflet of the plasma membrane. Annexin V has a great
affinity for PS and annexin V FITC staining is a good measure of PS
translocation (35). As shown in Figure 2,
granulysin induces increased annexin V staining. We previously showed
that granulysin also induces cell death of JY (a B lymphoblastoid cell
line), K562, and YAC (17). Treatment of these cell lines with
granulysin similarly increases annexin V staining (data not shown).
Hence, granulysin treatment of cells causes cell death with the
characteristics of apoptosis.
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The effect of granulysin on ceramide and sphingomyelin levels in
target cells was examined. Jurkat cells treated with 50 µM granulysin
for 16 h were killed, as determined by the MTT assay and trypan
blue staining, while cells treated with recombinant HSC-70 protein were
not (Fig. 3c). Granulysin
induced a significant increase in cellular ceramide content and a
decrease in that of sphingomyelin (Fig. 3a, lane
3), while no changes in sphingomyelin and ceramide levels were
observed in HSC-70-treated cells (Fig. 3a, lane
2). The Cer/SM ratio was increased 6-fold in granulysin-treated
cells (Fig. 3b). By comparison, anti-Fas mAb-induced
apoptosis, which is associated with sphingomyelinase activation with a
similar time course, increased the Cer/SM ratio by 2.6-fold (26) (data
not shown). Thus, granulysin-induced apoptosis is associated with
sphingomyelinase activation and ceramide generation.
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Ceramide has been proposed as a mediator of some apoptotic
processes (23, 24), and it induces apoptosis when added exogenously to
cells (25, 26). To study the involvement of ceramide in
granulysin-induced apoptosis, Jurkat cells were cultured for 72 h
in the presence of 50 µM fumonisin B1, a potent inhibitor
of ceramide synthase. This treatment resulted in almost complete
depletion of sphingomyelin and ceramide (Fig. 4a). Ceramide levels did not
increase in fumonisin B1-treated cells incubated with
either anti-Fas mAb (40) or granulysin (data not shown). However,
granulysin-induced Jurkat cell death was similar in cells treated or
not with the ceramide synthase inhibitor (Fig. 4b).
Granulysin induced chromatin condensation and nuclear fragmentation in
fumonisin B1-treated cells (Fig. 4c) similarly
to that observed in cells not treated with the ceramide synthase
inhibitor (see Fig. 1
C).
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Intracellular cysteine proteases with Asp specificity (caspases)
are the main executioners of apoptosis (41, 42). We previously reported
that Ac-DEVD-CHO, a tetrapeptide inhibitor of the CPP32-like subfamily
of caspases, completely inhibits Fas-induced apoptosis in Jurkat cells,
but does not affect ceramide-induced cell death (26). CPP32, like other
members of the caspase family, is activated by proteolytic processing
(38). Activation of CPP32 can be analyzed by Western blotting with a
specific mAb by monitoring the disappearance of the 32-kDa proenzyme.
Treatment of Jurkat cells with anti-Fas mAb resulted in
strong activation of CPP32 (Fig. 5a, lane 2), while
treatment with exogenous ceramide caused only minor reduction in the
levels of the proenzyme (Fig. 5a, lane 3). While
Fas-induced apoptosis was completely blocked by the peptide inhibitor
Ac-DEVD-CHO, apoptosis of ceramide-treated Jurkat cells was unaffected
by the same concentration of inhibitor, indicating that neither CPP32
nor other CPP32-like caspases are implicated in ceramide-induced
apoptosis (Fig. 5, b and c).
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, b and
c). Thus, in Jurkat cells, ceramide-induced apoptosis is
mediated by the activation of one or several Z-VAD-sensitive,
DEVD-insensitive caspases. Effect of peptide caspase inhibitors on granulysin-induced apoptosis
The effect of granulysin treatment on CPP32 activation was tested
by immunoblot, as indicated above for ceramide and anti-Fas mAb. As
shown in Figure 6a, CPP32
processing as a consequence of granulysin treatment was very limited,
similar to that observed in ceramide-treated cells (see Fig. 5a, lane 3). The effect of peptide caspase
inhibitors on granulysin-induced apoptosis was also tested. Apoptosis
of Jurkat cells treated with granulysin was only slightly decreased by
Ac-DEVD-CHO, whereas Z-VAD-fmk significantly protected Jurkat cells
from granulysin-induced cell death (60% of protection, Fig. 6, b and c). This pattern was similar to that
observed for ceramide-induced apoptosis (see Fig. 5) and could reflect
the contribution of ceramide generation induced by granulysin (Fig. 3)
to cell death. However, there is a significant (40%) Z-VAD-resistant
component in granulysin-induced apoptosis. This component appears to be
ceramide independent and may correspond to the mechanism involved in
cytolysis of fumonisin B1-treated cells (Fig. 4). In fact,
while Z-VAD-fmk partially inhibited granulysin toxicity in Jurkat cells
(Fig. 6), it had no effect on granulysin-induced apoptosis in ceramide-
and SM-depleted Jurkat cells (Fig. 7).
The granulysin toxicity on fumonisin B1-treated cells was
similar to that in normal Jurkat cells, in agreement with the results
shown in Figure 4.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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By homology with pore-forming proteins, such as amoebapores and perforin, granulysin could interact with lipids in the plasma membrane, forming pores. Two hypothetical models could then explain granulysin-induced sphingomyelinase activation: 1) in a manner similar to that proposed for saposin A in the activation of acidic sphingomyelinase (21), granulysin could activate plasma membrane neutral sphingomyelinase at the same time as granulysin forms pores in the membrane; or 2) as described for all saposins, granulysin could activate acidic sphingomyelinase (20, 21, 48). Since acid sphingomyelinase is restricted to the lysosomal compartment, the second possibility would implicate granulysin internalization and localization into this compartment, a more complex process than mere insertion into the plasma membrane. Additional studies are needed to characterize the mechanism of action of granulysin, especially regarding its subcellular localization while inducing its toxic effect and its potential for pore formation.
The marked (sixfold) increase in the Cer/SM ratio associated with granulysin toxicity appears to implicate ceramide generation as a mediator of granulysin-induced apoptosis. Nevertheless, granulysin was able to exert a toxic effect in cells depleted of sphingomyelin and ceramide by prolonged culture in the presence of the ceramide synthase inhibitor fumonisin B1. Apoptosis induced by exogenous ceramide was inhibited by Z-VAD-fmk (this work and 49 , but not by Ac-DEVD-CHO (this work and 26 . Z-VAD-fmk inhibits the proteolytic activity of several caspases (41, 43), while Ac-DEVD-CHO preferentially inhibits CPP32 and related caspases (41). Z-VAD-fmk inhibits the processing and activation of CPP32, at least during Fas-induced apoptosis (43), presumably by inhibiting FLICE (caspase-8) (44). Together these data suggest that ceramide-induced apoptosis is dependent on a Z-VAD-sensitive, DEVD-insensitive caspase. Caspases 1, 4, 5, and 6 exhibit similar sensitivities to these peptides (41, 50), although only activated CPP32 (caspase-3) and Mch2 (caspase-6) have been detected in apoptotic Jurkat cells (51).
Granulysin-induced cell death had an inhibitory profile similar to that induced by exogenous ceramide: weak protection by Ac-DEVD-CHO and strong protection by Z-VAD-fmk. These data suggest that the observed increase in cellular ceramide content as a consequence of granulysin treatment is indeed an important part of granulysin toxicity. However, there is also a Z-VAD-fmk-resistant component (>40%) in granulysin-induced apoptosis that is not observed when cell death is induced by exogenous ceramide. This component should be ceramide independent and could correspond to the lytic mechanism that mediates the entire granulysin toxic effect in SM-depleted cells. In fact, the observation that Z-VAD-fmk had no effect on granulysin toxicity in SM-depleted cells indicates that the ceramide-dependent and the Z-VAD-sensitive components are equivalent in granulysin-induced apoptosis.
The ceramide-independent granulysin-induced lytic pathway may be associated with the formation of membrane pores, although further studies are needed to demonstrate this possibility. In this respect, the dose-response toxicity obtained with Jurkat cells is similar to that previously obtained for granulysin-induced 51Cr release in YAC cells, suggesting that apoptosis, pore formation, and 51Cr release occur at the same doses of granulysin. The ceramide-independent component of granulysin-induced lysis is also apoptotic, associated with chromatin condensation and nuclear fragmentation, as demonstrated in SM-depleted cells. This effect could be associated with the increase in intracellular Ca+2 concentration induced by pores in the plasma membrane, since apoptotic cell death can be induced by calcium ionophores alone (52, 53). However, perforin-induced lysis has not been associated with nuclear fragmentation in some studies despite the formation of pores in the plasma membrane (45, 46), and ceramide-independent apoptotic cell death induced by granulysin could be dependent on some uncharacterized particular property of this protein.
In summary, we provide the first evidence for the biochemical mechanism of granulysin-induced lysis. Granulysin treatment of cells induces sphingomyelin-derived ceramide generation and the activation of Z-VAD-sensitive, DEVD-insensitive caspases, leading to apoptosis. This may constitute an additional lytic mechanism used by CTL and NK cells to eliminate virus-infected and tumor cells. However, granulysin-induced apoptosis also takes place through ceramide- and caspase-independent mechanisms that remain to be elucidated.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Alan M. Krensky, Division of Immunology and Transplantation Biology, Department of Pediatrics, Stanford University School of Medicine, Stanford, CA 94305-5208. E-mail address:
3 Abbreviations used in this paper: MTT, 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide; Ac-DEVD-CHO, N-acetyl-Asp-Glu-Val-Asp aldehyde; Z-VAD-fmk, benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone; CPP32, cysteine protease p32 (caspase 3); PS, phosphatidylserine; Cer/SM, ceramide/sphingomyelin ratio; PPDA, p-phenylenediamine.
Received for publication December 15, 1997. Accepted for publication April 16, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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tze, T. Machleidt, D. Witte, M. Krönke. 1994. Functional dichotomy of neutral and acidic sphingomyelinases in tumor necrosis factor signaling. Cell 78:1005.[Medline]
1. Biochem. J. 284:189.
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) or the presence of 600 µM DEVD-CHO (
) or 100 µM
Z-VAD-fmk (
). C18-ceramide (200 nM) dissolved in
ethanol/dodecane (98/2, v/v) or 20 ng/ml of anti-Fas mAb was then
added, as indicated, and cells were incubated for another 16 h.
The final concentration of ethanol/dodecane was 0.4% (v/v). Cell
viability was determined by the MTT assay (b), and cell
death was quantitated by trypan blue exclusion method
(c). Results are the mean ± SD of quadruplicate
determinations in two different experiments.
). Granulysin (50 µM)
was then added, and cells were incubated for an additional 16 h.
Cell viability was determined by the MTT assay (b), and
cell death was quantitated by trypan blue exclusion (c).
Results are the mean ± SD of quadruplicate determinations in two
different experiments.