| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
*
Division of Immunology and Transplantation Biology, Department of Pediatrics, and Departments of
Cardiothoracic Surgery and
Molecular Pharmacology, Stanford University School of Medicine, Stanford, CA 94305;
Department of Pediatrics, University of California, San Francisco, CA 94143;
¶
Gwen Knapp Center for Lupus and Immunology Research, Howard Hughes Medical Institute, University of Chicago, Chicago, IL 60637;
||
Division of Cellular Immunology, La Jolla Institute for Allergy and Immunology, San Diego, CA 92121;
#
Department of Medicine, University of Pittsburgh, Pittsburgh, PA 15213;
**
Evanston Northwestern Research Institute, Northwestern University, Evanston, IL 60201;
Abramson Family Cancer Research Institute, University of Pennsylvania, Philadelphia, PA 19104; and
*
Department of Bioquimica, Facultad de Ciencias, Universidad de Zaragoza, Zaragoza, Spain
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
) in mitochondria, and causes release of
cytochrome c. Granulysin-induced apoptosis is blocked in
cells overexpressing Bcl-2. Despite the release of cytochrome
c, procaspase 9 is not processed. Nevertheless,
activation of caspase 3 is observed in granulysin-treated cells,
suggesting that granulysin activates a novel pathway of CTL- and NK
cell-mediated death distinct from granzyme- and death receptor-induced
apoptosis. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
CTL kill targets in part by granule exocytosis (4). Granule-mediated cytotoxicity involves the action of the pore-forming protein perforin and a family of serine proteases, the granzymes. Although granzyme B can cleave many caspase substrates in vitro (5, 6), it was recently demonstrated that granzyme B-induced apoptosis results from its ability to directly cleave BH3-interacting domain death agonist (Bid)4 (7), a Bcl-2 family member, which then transmits the death signal through the mitochondrion. Granzyme A causes apoptosis through a caspase-independent pathway (8). Cell-mediated cytotoxicity can also involve ligation of death receptors on the target cell using a second signaling pathway for initiating caspase-dependent apoptosis (9, 10). Granulysin is an additional secreted protein present in cytolytic granules (1).
We previously showed that recombinant granulysin, independent of other granule constituents, induces apoptotic cell death of Jurkat tumor cells (11). Target cells show the physical hallmarks of apoptosis, including nuclear condensation, membrane ruffling, and translocation of phosphatidylserine to the outer leaflet of the plasma membrane, as detected by annexin V-FITC staining. Granulysin activates a sphingomyelinase in target cells, increasing the cellular ceramide/sphingomyelin ratio. We also showed that a general inhibitor of caspases, the peptide benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone, partially protects against granulysin-induced cell death, whereas a more specific inhibitor of caspase 3-like enzymes, the peptide N-acetyl-Asp-Glu-Val-Asp aldehyde, does not protect.
Granulysin is homologous to the saposin-like protein (SAPLIP) family of
lipid-binding proteins (1). The homology of granulysin to
saposins suggests that it may exert its cytolytic function and
antibacterial activity via interaction with lipids. In this study, we
demonstrate that granulysin disrupts negatively charged synthetic
liposomes and cell membranes. Cells expressing the antiapoptotic
gene Bcl-2 are protected against granulysin-induced
cell death. The interaction of granulysin with cells causes a change in
mitochondrial potential () and release of cytochrome
c. Surprisingly, however, procaspase 9 is not processed to
its active form, although an early caspase 3 activation is observed.
Thus, granulysin is a CTL- and NK cell-specific product that activates
a death pathway distinct from those induced by Fas or granzymes.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Reagents used include chemicals (Sigma, St. Louis, MO), propidium iodide (Molecular Probes, Eugene, OR), lipids (Avanti Polar Lipids, Alabaster, AL), fluorescent probes 1-aminonaphtlene-3,6,8-trisulfonic acid, and N,N'-(p-phenylenedimethylene)bis(pridinium bromide), and fura 2 (Molecular Probes), HRP-conjugated secondary Abs (Cappell, Durham, NC), and ECL reagents (Amersham Pharmacia Biotech, Piscataway, NJ).
Cells
The Jurkat T cell leukemia (American Type Culture Collection, Manassas, VA) was cultured as described (11). Jurkat cells transfected with Bcl-2 or cytokine response modifier (CrmA) were maintained in medium supplemented with 0.5 mg/ml G418, as described (12). Jurkat cells overexpressing Toso were sorted by flow cytometry selecting for green-fluorescent protein expression from the viral construct used for infection (13). FL5.12 cells were grown as described (14).
Expression and purification of recombinant granulysin and purification of perforin
Recombinant granulysin was expressed and purified as described (1). Briefly, recombinant 9-kDa granulysin was expressed using the vector pET28a (Novagen, Madison, WI). Fusion protein was purified in 6 M guanidine HCl on nickel affinity resin. After refolding and dialysis, the histidine tag was removed by thrombin cleavage, and the material was further purified by reverse-phase HPLC. After lyophilization, the protein was suspended in PBS, and concentration was determined using a protein assay (Bio-Rad, Hercules, CA) with lysozyme as a standard. Human perforin was purified by metal-affinity chromatography from the human NK cell line YT2C2, as described (6).
Liposome lysis assays
Disruption of liposomes was measured as described (15). Briefly, the fluorescent probe 1-aminonaphtlene-3,6,8-trisulfonic acid and its collisional quencher N,N'-(p-phenylenedimethylene)bis(pridinium bromide) were incorporated into palmitoyloleoylphosphatidylglycerol (POPG) and palmitoyloleoylphosphatidylcholine (POPC) large unilamellar vesicles. POPC and cardiolipin (CL) were mixed in a 1:1 ratio to form large unilamellar vesicles. Release of the fluorescent probe was measured on a spectrophotometer. Liposomes (25 µM phospholipids) were treated with 550 nM granulysin, and the percentage of specific lysis was calculated.
Cytotoxicity assays
Jurkat cells were seeded in 96-well plates at a density of 2 x 105/ml (100 µl/well) in medium containing 0.1% FCS. Cells were cultured for 16 h in the presence of medium, granulysin (11), or anti-Fas mAb (CH-11, 100 ng/ml; Beckman-Coulter, Fullerton, CA). Cell death was measured by propidium iodide uptake and by gating of live cells based on forward vs side scatter. Similar results with Jurkat cells were obtained using additional cytotoxicity assays, including 51Cr release (1), MTT assay (16), and annexin V-FITC staining (11). Plasma membrane integrity was measured by propidium iodide uptake after 15 min of culture in the presence of granulysin.
Measurement of mitochondrial membrane potential with Rho-123
Jurkat cells were washed, resuspended at 5 x 105/ml in RPMI 1640 supplemented with 0.1% FCS, and incubated for 1 h at 37°C. A total of 105 cells were treated with recombinant granulysin for 2 h at 37°C, washed, resuspended in PBS, and placed on an inverted coverslip chamber (pretreated with polylysine). After 5 min, 200 µl of 20 µM Rho-123 (Molecular Probes) was added, and the excess was washed away after 2 min. Confocal images were collected using a Molecular Dynamics (Sunnyvale, CA) MultiProbe 2010 Confocal Laser Scanning Microscope, with a Nikon Diaphot 200 inverted with epi-fluorescence attachment. A krypton/argon laser with 488-nm excitation and a 590-nm long pass emission filter was used to collect images through a x60 (oil) objective. After images had been collected, 200 µl of 2 µM carbonyl cyanide m-chlorophenylhydrazone (CCCP; Sigma) was added to the cells in the chamber, and, after 30 s, additional images were collected. Results are representative of 10 fields for each sample.
Ca2+ flux measurements
Jurkat cells were loaded with fura 2 in a 1:1 vol of cell
suspension with dye by incubating at 37°C for 60 min. The cells were
then washed twice with buffer and incubated for a further 30 min for
complete deesterification. The induction of a calcium flux was observed
using an Attofluor digital fluorescence microscope (Atto Instruments,
Rockville, MD). Cell suspension was loaded into chambers and,
after 
20 s, these cells were treated with ionomycin or granulysin.
The Ca2+-dependent fluorescence signal was
obtained using excitation at 340 and 380 nm and ratioing the
fluorescence intensities detected at 510 nm.
Subcellular fractionation and Western blot analysis
Mitochondrial and cytosolic (S100) fractions were prepared by resuspending FL5.12 cells in 0.8 ml ice-cold buffer A (250 mM sucrose, 20 mM HEPES, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 17 µg/ml PMSF, 8 µg/ml aprotinin, 2 µg/ml leupeptin (pH 7.4)). Cells were homogenized using a prechilled cylinder cell homogenizer (H&Y Enterprise, Redwood City, CA). Unlysed cells and nuclei were pelleted at 750 x g for 10 min, and the supernatant was centrifuged at 10,000 x g for 25 min. The pellet, representing the mitochondrial fraction, was resuspended in buffer A, and the supernatant was subjected to further centrifugation at 100,000 x g for 1 h. The supernatant from the final centrifugation represents the S100 fraction. Equivalent amounts of mitochondrial and S100 fractions were subjected to Western blot analysis, as previously described (14). The primary Abs were either a 1/1000 dilution of the 7H8.2C12 cytochrome c mAb (a gift from R. Jemmerson, Department of Microbiology and Center for Immunology, University of Minnesota, Minneapolis, MN) in TBS, 0.2% Tween 20; or 10 ng/ml 20E8-C12 cytochrome oxidase subunit IV mAb (Molecular Probes) as a control for mitochondrial inner membrane integrity.
For caspases 3 and 9, Jurkat cells were treated with 50 µM granulysin
for 3 h and washed, and whole-cell lysates were prepared by lysing
cells in 2% SDS, 137 mM NaCl, 2.7 mM KCl, and 8 mM
NaHPO4 (pH 7.4) (17, 18). Samples
were separated on 15% SDS-PAGE and electroblotted to polyvinylidene
difluoride membranes. Membranes were blocked in 5% nonfat milk, 0.1%
Tween 20 in TBS and then incubated with specific Abs to caspase 9 (Cell
Signaling Technologies, Beverly, MA) or caspase 3 (BD PharMingen, San
Diego, CA) and 
-actin (Calbiochem, La Jolla, CA). Primary Abs were
detected by peroxidase-labeled secondary Ab and developed using ECL
(Amersham Pharmacia Biotech) and exposure to film.
Xenopus egg extracts and incubations
Recombinant truncated human Bid (tBid) was produced as previously described (19). Xenopus egg mitochondria were isolated as previously described (20). Mitochondrial incubations were reconstituted from mitochondria (1 mg/ml; biuret method) in buffer A (250 mM sucrose, 20 mM HEPES/KOH (pH 7.5), 100 mM KCl, 2.5 mM MgCl2, 1 mM DTT, 5 µg/ml cytochalasin B, and 50 µg cycloheximide) and an ATP-regenerating system of 10 mM phosphocreatine, 2 mM ATP, and 150 µg/ml creatine phosphokinase. To examine mitochondrial cytochrome c content, extract aliquots (10 µl) were removed at 3 h and mixed with 0.5 ml buffer A before centrifugation (12,000 x g, 5 min) to pellet mitochondria. Western blot of mitochondrial cytochrome c was performed as described previously (19).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Granulysin, a lytic molecule of human CTL and NK cell granules, is
homologous to the SAPLIP family of lipid-binding proteins
(1). The homology of granulysin to saposins suggests that
it may exert its lytic activity via interaction with membrane lipids.
To assess granulysin interaction with lipids, the lytic effect of
granulysin on synthetic liposomes of defined composition was examined
(Fig. 1
A). Negatively charged
POPG liposomes were disrupted, whereas neutral POPC liposomes were not.
Liposomes consisting of a mixture of POPC and CL, a negatively charged
lipid, were disrupted by granulysin, indicating that lysis was
associated with negatively charged lipids. Disruption of liposomes by
granulysin was dependent on both granulysin (Fig. 1B) and
lipid concentration (Fig. 1C). In these experiments, we
reproducibly detect significant disruption of liposomes at granulysin
concentrations as low as 0.1 µM. Thus, granulysin, like other SAPLIP
family members, interacts with lipid membranes.
|
). Perforin is shown as a positive
control for membrane disruption. The combination of granulysin and
perforin was additive rather than synergistic, indicating that these
two molecules act independently to induce membrane damage (data not
shown). Thus, granulysin perturbs the plasma membrane by a direct
interaction with lipids. This activity is accompanied by nuclear
condensation, membrane ruffling, and annexin V-FITC staining, as
previously described (11).
|
Bcl-2 protects cells from the mitochondrial apoptotic pathway
(21), whereas CrmA protects cells from death
receptor-induced apoptosis by inhibiting several caspases
(22). Jurkat cells transfected with the genes for
Bcl-2 or CrmA (12) were protected to some extent against
granulysin-induced death relative to vector controls (Fig. 3). Bcl-2 protection indicates that the
apoptotic pathway induced by granulysin involves mitochondrial damage
(21), whereas CrmA protection further implicates caspases
in the death pathway (22).
|
) (Fig. 4). Rho-123 is a cationic fluorophore
that is taken up and concentrated in electronegative mitochondria in
response to the . CCCP is a protonophore that disrupts the
, leading to the dissipation of the dye into the cytoplasm
(23, 24, 25). Jurkat cells incubated in medium plus Rho-123
show a punctate fluorescence (Fig. 4A) that is abolished in
cells after the addition of CCCP (Fig. 4B).
Granulysin-treated cells do not stain with Rho-123 (Fig. 4C), and addition of CCCP does not cause any change in the
staining (Fig. 4D). Similar results were obtained with DiOC6
(3) and JC-1, two other potential sensitive dyes (not
shown).
|
A), implicating
granulysin-induced cytochrome c release as an initiating
signal for caspase activation.
|
B),
indicating that the mitochondrial disruption observed in cells is not a
direct effect of pure granulysin.
One indirect mechanism by which granulysin-induced plasma membrane
damage could affect mitochondria is through an influx of ions across
the plasma membrane. Granulysin could induce an influx of
Ca2+ across the plasma membrane, which would
induce permeability transition, secondary swelling of mitochondria,
rupture, and release of cytochrome c. Chelating divalent
cations with EDTA partially inhibits granulysin-induced death in
chromium release assays of labeled target cells (Fig. 6A), but does not affect the
ability of granulysin to disrupt cell membranes (Fig. 6B) or
liposomes (data not shown). The calcium flux induced by granulysin was
directly detected by the use of the dye fura 2. Jurkat cells loaded
with fura 2 and treated with either ionomycin or granulysin show a
rapid increase in fluorescence, indicative of a calcium flux into the
cytosol (Fig. 6C). Thus, there is a requirement for
extracellular ions in granulysin-induced apoptosis, and an ion flux
induced by disruption of the plasma membrane may initiate cell death.
Additionally, these data show that unlike perforin, there is not a
requirement for calcium in granulysin-induced membrane damage,
indicating a distinct mechanism for granulysin-induced plasma membrane
disruption.
|
Granulysin-induced apoptosis involves caspases, as both CrmA and benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone partially protect target cells. To identify specific caspases involved in granulysin-induced death, we examined three initiator caspases involved in Fas-, granzyme B-, and genotoxic-mediated apoptosis.
The cowpox serpin CrmA inhibits death pathways mediated by Fas and
chemotherapeutic agents (27). We observed that CrmA, an
inhibitor of serine and cysteine proteases, including caspase 8
(28), protects from granulysin-induced death (Fig. 3).
Because the Jurkat-Bcl-2 transfectant is resistant to granulysin but is
susceptible to Fas-induced death, it is unlikely that the defined death
receptor pathway is involved in granulysin-induced apoptosis. To
further evaluate the role of caspase 8 in the granulysin-mediated
pathway, we tested the ability of another inhibitor to protect Jurkat
cells from granulysin-induced death. Nolan and coworkers
(13) identified a molecule designated Toso that inhibits
Fas- and Fas-associated death domain protein-induced apoptosis. Toso
prevents cell death by inducing the expression of cellular
Fas-associated death domain-like IL-1-converting enzyme inhibitory
protein (29) and blocking adapter-induced caspase 8
activation (30). Jurkat cells expressing Toso were
protected from anti-Fas-induced death, but were not protected from
granulysin-induced death (data not shown), again indicating that
granulysin-induced death is different from the death receptor signaling
pathway mediated by Fas.
Caspase 3 is a substrate for granzyme B (5, 6).
Previously, we reported minimal processing of procaspase 3 in
granulysin-treated cells and that N-acetyl-Asp-Glu-Val-Asp
aldehyde, a peptide inhibitor of caspase 3-like enzymes, only weakly
protected against granulysin-induced apoptosis of Jurkat cells
(11). In those experiments, cells were incubated with
granulysin for 16 h before measuring apoptosis or procaspase 3
cleavage. Because granulysin-mediated damage is detectable much
earlier, we decided to assay procaspase 3 activation at earlier times.
As shown in Fig. 7, procaspase 3 is
processed to some extent in cells treated for 3 h with 50 µM
granulysin or 2 µM staurosporin.
|
). Processing of
caspase 9 in staurosporine-treated cells is apparent. However,
processed forms of caspase 9 were not detected in granulysin-treated
cells. Thus, granulysin induces the activation of caspase 3 by a
mechanism that is independent of processing of caspase 9. |
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
In vitro, we find that granulysin strongly induces apoptosis of tumor target cells at a concentration of 50 µM. This relatively high concentration is consistent with granulysin-inducing apoptosis in a receptor-independent manner (33). However, it is unlikely that such high concentrations of granulysin are necessary to effect target cell lysis in vivo. In vivo, granulysin would be delivered in a relatively concentrated form at the junction of the CTL/target (4). Furthermore, granulysin most likely works in concert with other constituents of cytolytic granules, as previously shown for both granzymes and perforin. Finally, conditions in the local environment will also affect granulysin-mediated lysis, because protein, salt, and H+ can alter the lytic activity of recombinant granulysin (34).
An immediate effect of granulysin on target cells is disruption of plasma membrane integrity. Positively charged granulysin lyses negatively charged, but not neutral, liposomes. NK-lysin, the SAPLIP family member with the greatest homology to granulysin (35), also affects lipid membranes (36). NK-lysin disrupts lipid vesicles and induces random current fluctuations through a planar lipid membrane when voltage is applied across the membrane, indicating that the lipid membrane-disrupting activity of NK-lysin does not involve formation of a stable pore (36). Our findings suggest a similar mechanism of action for granulysin. Unlike the pore-forming molecule perforin (37), granulysin-induced membrane damage leads to activation of the apoptotic machinery. Granulysin disruption of the plasma membrane leads to a calcium flux that may in turn damage mitochondria. Mitochondrial damage is apparent within minutes of cell surface exposure to granulysin, but this effect is indirect in that recombinant granulysin is incapable of directly damaging isolated mitochondria in vitro. The inability of granulysin to directly disrupt the mitochondrial membrane may be either due to differences between the lipid compositions of the plasma membrane and mitochondrial membrane or that an intermediate is involved in transmitting the granulysin signal. One candidate for such an intermediate is an ion flux. We show that EDTA partially inhibits granulysin-induced death in chromium release assays, but it has no effect on the membranolytic properties of granulysin. Furthermore, there is a rapid rise in intracellular Ca2+ associated with granulysin treatment of cells, suggesting that plasma membrane damage may provoke mitochondrial damage and cytochrome c release.
Previous studies with caspase inhibitors indicated that caspase activation is involved in granulysin-mediated cell death (11). In the present study, we further evaluated the role of caspases in the granulysin pathway. A transfectant overexpressing the serpin CrmA protected against granulysin-mediated cell death. CrmA is reported to preferentially inhibit caspase 8 and caspase 1 at the concentrations generally present in transfections (28), but our experiments using Toso transfectants indicate that death receptors are not involved in granulysin-mediated cell death (13). Toso expression induces Fas-associated death domain-like IL-1-converting enzyme inhibitory protein expression, which acts to protect cells from Fas- and Fas-associated death domain protein-induced death (29). We find that, although Toso-expressing Jurkat are protected from anti-Fas, they are not protected from granulysin. Thus, granulysin-induced death is not dependent on the death receptor pathway mediated by Fas.
Consistent with this conclusion, Jurkat cells overexpressing Bcl-2
showed only weak protection from anti-Fas-induced death (Fig. 3),
indicating that our Jurkat cell line is not a type II cell in which the
death receptor and mitochondrial death pathways intersect
(38). As these cells are protected from granulysin-induced
death, the granulysin pathway is clearly distinct from the death
receptor pathway mediated by Fas.
The antiapoptotic effect of Bcl-2 on granulysin-induced apoptosis
suggests that mitochondrial damage is a critical step in
granulysin-induced apoptosis (21). Apaf-1, the mammalian
ced-4 homologue, binds cytosolic cytochrome c and
recruits procaspase 9, leading to activation. Although we are unable to
detect processing of procaspase 9 in granulysin-treated cells, it has
been reported that unprocessed procaspase 9, when complexed with
cytochrome c and dATP, can activate downstream caspases
(39). It is also possible that low levels of processed
caspase 9, sufficient to cause apoptosis but undetectable by
immunoblot, may be present in granulysin-treated cells. Alternatively,
activation of caspase 3 may occur by a caspase 9-independent mechanism.
Disruption of in mitochondria releases not only cytochrome
c, but also a number of other molecules, including
apoptosis-inducing factor (AIF). AIF is sufficient to activate
endonucleases in isolated nuclei (40, 41, 42). Anel and
coworkers have recently demonstrated that AIF is released from
mitochondria in cells treated with
granulysin.5
A large number of loss of function studies performed by many laboratories over the last 5 years have clearly delineated the roles of perforin/granzymes and Fas systems for CTL-mediated cytotoxicity (43, 44). It is clear from these papers that most of the measurable in vitro cytotoxicity delivered by CTL in the reported systems can be attributed to these two pathways. This calls into question the biologic function of granulysin in other than an accessory role. We have been unable to identify a murine homologue for granulysin using a variety of approaches. Granulysin-specific antisera fail to Western blot a protein from mouse CTL extracts; no signal is obtained using several murine CTL/NK cell cDNA or genomic libraries probed with radiolabeled granulysin cDNA; and no murine homologue is present in databases. This observation is reminiscent of other antimicrobial peptides such as human defensins for which there are no structural murine homologues (45). Thus, granulysin appears not to have a mouse homologue that could conceivably complement the deficiency of perforin or granzyme B in knockout mice. Murine systems, including knockouts, cannot conclusively determine the functional role for granulysin- or other species-specific proteins in humans. It should also be noted that the various reported systems only address limited biologic functions. For example, granulysin may be important in local environments such as the gut or CNS. Although no granulysin knockout is possible in mice because there is no homologue, experiments with human granulysin transgenic mice are planned.
CTL evolved to induce target cell death via several pathways (4). Apoptosis is initiated by the delivery of granzymes through granule exocytosis and by stimulation of cell surface death receptors (46). Granzyme B activates several substrates, including caspase 3 (5, 6), whereas death receptor pathways of the TNF family, like Fas, use caspase 8 as the initiator (9). In this study, using a variety of approaches, we show that granulysin disrupts cell membranes, damages mitochondria, and causes release of cytochrome c and other factors (21). Thus, granulysin, a member of the SAPLIP family, acts via a distinct molecular mechanism for induction of target cell death by CTL and NK cells.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 A.A.K. and S.O. contributed equally to this work.
3 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 94305-5164. E-mail address: krensky{at}stanford.edu
4 Abbreviations used in this paper: Bid, BH3-interacting domain death agonist;SAPLIP, saposin-like protein; AIF, apoptosis-inducing factor; CCCP, carbonyl cyanide m-chlorophenylhydrazone; CL, cardiolipin; CrmA, cytokine response modifier; POPC, palmitoyloleoylphosphatidylcholine; POPG, palmitoyloleoylphosphatidylglycerol; Rho-123, Rhodamine-123; tBid, truncated human Bid.
5 J. Pardo, P. Pérez-Galán, S. Gamen, I. Marzo, I. Monleón, A. Kaspar, S. A. Susín, G. Kroemer, A. M. Krensky, J. Naval, and A. Anel. A role of the mitochondrial apoptosis-inducing factor (AIF) in granulysin-induced apoptosis. Submitted for publication.
Received for publication June 9, 2000. Accepted for publication April 16, 2001.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
converting enzyme. Cell 69:597.[Medline]
This article has been cited by other articles:
|
|
 |
|
  A. Nakashima, A. Shiozaki, S. Myojo, M. Ito, M. Tatematsu, M. Sakai, Y. Takamori, K. Ogawa, K. Nagata, and S. Saito Granulysin Produced by Uterine Natural Killer Cells Induces Apoptosis of Extravillous Trophoblasts in Spontaneous Abortion Am. J. Pathol., September 1, 2008; 173(3): 653 - 664. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. P. Huang, S.-C. Lyu, C. Clayberger, and A. M. Krensky Granulysin-Mediated Tumor Rejection in Transgenic Mice J. Immunol., January 1, 2007; 178(1): 77 - 84. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Liu, S. Liu, X. Qu, and J. Liu Construction of a eukaryotic expression system for granulysin and its protective effect in mice infected with Mycobacterium tuberculosis. J. Med. Microbiol., October 1, 2006; 55(Pt 10): 1389 - 1393. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Deng, S. Chen, Q. Li, S.-c. Lyu, C. Clayberger, and A. M. Krensky Granulysin, a Cytolytic Molecule, Is Also a Chemoattractant and Proinflammatory Activator J. Immunol., May 1, 2005; 174(9): 5243 - 5248. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Dong, Q. Li, S.-c. Lyu, A. M. Krensky, and C. Clayberger A novel apoptosis pathway activated by the carboxyl terminus of p21 Blood, February 1, 2005; 105(3): 1187 - 1194. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Q. Li, C. Dong, A. Deng, M. Katsumata, A. Nakadai, T. Kawada, S. Okada, C. Clayberger, and A. M. Krensky Hemolysis of Erythrocytes by Granulysin-Derived Peptides but Not by Granulysin Antimicrob. Agents Chemother., January 1, 2005; 49(1): 388 - 397. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. L. Ma, C. L. C. Wang, G. G. Neely, S. Epelman, A. M. Krensky, and C. H. Mody NK Cells Use Perforin Rather than Granulysin for Anticryptococcal Activity J. Immunol., September 1, 2004; 173(5): 3357 - 3365. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Okada, Q. Li, J. C. Whitin, C. Clayberger, and A. M. Krensky Intracellular Mediators of Granulysin-Induced Cell Death J. Immunol., September 1, 2003; 171(5): 2556 - 2562. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Bose, Y. Inoue, K. E. Kokko, and F. G. Lakkis Cutting Edge: Perforin Down-Regulates CD4 and CD8 T Cell-Mediated Immune Responses to a Transplanted Organ J. Immunol., February 15, 2003; 170(4): 1611 - 1614. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. L. Ma, J. C. L. Spurrell, J. F. Wang, G. G. Neely, S. Epelman, A. M. Krensky, and C. H. Mody CD8 T Cell-Mediated Killing of Cryptococcus neoformans Requires Granulysin and Is Dependent on CD4 T Cells and IL-15 J. Immunol., November 15, 2002; 169(10): 5787 - 5795. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Q. Sun, R. L. Burton, and K. G. Lucas Cytokine production and cytolytic mechanism of CD4+ cytotoxic T lymphocytes in ex vivo expanded therapeutic Epstein-Barr virus-specific T-cell cultures Blood, May 1, 2002; 99(9): 3302 - 3309. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Pardo, P. Perez-Galan, S. Gamen, I. Marzo, I. Monleon, A. A. Kaspar, S. A. Susin, G. Kroemer, A. M. Krensky, J. Naval, et al. A Role of the Mitochondrial Apoptosis-Inducing Factor in Granulysin-Induced Apoptosis J. Immunol., August 1, 2001; 167(3): 1222 - 1229. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||
) or surfactant protein B
(
) on liposomes of different compositions. Granulysin (0.5 µM) or
surfactant protein B was added to 25 µM liposomes, and the percentage
of specific release was measured. B, Dose-dependent
disruption of liposomes by granulysin. A total of 25 µM POPG or
POPC-CL liposomes were treated with increasing amounts of granulysin.
C, Lipid concentration dependence of granulysin-induced
disruption of liposomes. Granulysin (0.5 µM) was added to cuvettes
containing increasing amounts of POPG or POPC-CL liposomes, and
specific release was measured. Data in each panel are from independent
experiments using separate liposome preparations. Data represent
duplicate experiments.
