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Experimental Immunology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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was also induced by both CTL
effector pathways, and this was also specifically blocked by caspase
inhibitors when induced by the FasL/Fas, but not by the granule
exocytosis, pathway. Similarly, target membrane blebbing induced by CTL
via the FasL/Fas, but not via the granule exocytosis, effector pathway
was specifically blocked by caspase inhibitors. In contrast to the
above nonnuclear damage, CTL-induced target staining by the lipid probe
FM1–43 reflecting plasma membrane endocytosis was blocked by caspase
inhibitors. Thus, when caspase activation is blocked, the granule
exocytosis pathway triggers several parameters of target apoptotic
damage in addition to lysis, suggesting that granzymes directly trigger
a postcaspase cytoplasmic apoptotic death
pathway. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Since target cells killed by cytotoxic lymphocytes in vitro typically display characteristic apoptotic features (5), an appealing hypothesis has been that a common apoptotic death pathway existing in a latent form in all cells is triggered by these effector cells (6). Rapid progress has been made recently in identifying a protease family termed caspases as central molecular mediators of apoptotic cell death (7). The 10 currently identified human caspases are widely expressed in an inactive precursor form in many cells and have the hallmark specificity of cleaving protein substrates with aspartic acid residues at the P1 position. Their activation occurs as a result of proteolytic processing of procaspases at aspartic acid residues, so that caspases can autoactivate and process each other in an activation cascade (8). Despite the general consensus that caspases play a central role in apoptotic death, important questions remain unanswered. One of these is the nature of postcaspase cell destruction pathway leading to the various characteristic features of apoptotic cell death, including phosphatidylserine (PS) externalization and lysis.
Recent experiments have shown that both effector pathways used by cytotoxic lymphocytes activate caspases. Cross-linking of target cell Fas by FasL on CTL membranes leads to activation of caspases 8 and probably 10 via the adaptor molecule FADD (9). These caspases can process and activate downstream caspases that, in turn, trigger cell destruction. We have shown that rapid target cell lysis and apoptotic nuclear damage by this CTL effector pathway are blocked by two classes of caspase inhibitors, confirming the predicted functional importance of caspases in this effector pathway (10).
For the granule exocytosis pathway, the requirement for caspases in target cell death is less clear. Caspase activation via the granule exocytosis pathway is predicted because the granule serine protease granzyme B recognizes a sequence motif compatible with caspase activation (11) and has been shown to initiate processing and activation of several caspases (12). CTL targets undergo rapid caspase-3 processing as predicted by these in vitro experiments (13), thus providing an explanation for apoptotic nuclear damage induced by granzyme B in the presence of sublytic doses of perforin (14). However, while CTL from mice lacking granzyme B induce target nuclear damage somewhat more slowly, their potency and rate of target lysis via the granule exocytosis pathway are unaffected by the loss of granzyme B (15). (Lysis by activated NK cells from mice lacking granzyme B is partially defective (16).) Since no other granule proteases are known to activate caspases directly, these data suggest that target lysis via CTL granule exocytosis might be independent of caspases. In support of this, we recently showed that two classes of caspase inhibitors that effectively blocked CTL granule exocytosis-induced target nuclear damage did not detectably block CTL-mediated target lysis (10).
One model to explain these results proposes that granzyme B mediates nuclear damage via caspase activation, but that target lysis occurs as a result of granzyme-induced cleavage of downstream cytoplasmic protein substrates which are also cleaved by caspases and lead to apoptotic cell destruction. This model predicts that other CTL granule exocytosis-induced cytoplasmic apoptotic damage might be caspase independent if it is part of the postcaspase death pathway. In this paper we describe experiments showing that three such nonnuclear changes induced by the granule exocytosis pathway in CTL and NK cells are not blocked by caspase inhibitors that block nuclear damage. These results suggest that granzymes activate a postcaspase apoptotic damage pathway that results in mitochondrial potential loss, PS surface exposure, membrane blebbing, and lysis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The protease inhibitors Cbz-Val-Asp(O-methyl)-fluoromethyl ketone (ZVAD-FMK), Boc-Asp(O-methyl)-fluoromethyl ketone (BD-FMK), and Cbz-Phe-Ala-fluoromethyl ketone (ZFA-FMK) were purchased from Enzyme Systems Products (Dublin, CA), made up as stock solutions of 50 mM in DMSO, and stored at -70°C. IgG anti-Fas mAb DX2 was obtained from PharMingen (San Diego, CA). FM1–43, DiIC16 (3), and DiOC6 (3) were obtained from Molecular Probes (Eugene, OR) made up as 1 mM stocks in DMSO, and stored at 4°C. Annexin V-FITC was obtained from Trevigen (Gaithersburg, MD). PMA, ionomycin, and Hoechst 33342 were obtained from Sigma (St. Louis, MO).
Target and effector cells
The human lymphoblastoid cell line Jurkat and the erythromyeloid leukemia K562 were maintained in complete medium (RPMI 1640 supplemented with 10% FCS, 100 IU penicillin, and 10 µg/ml streptomycin) and used as target cells. To distinguish these cells from effector cells by flow cytometry or fluorescence microscopy, they were prelabeled by incubation with the lipid probe DiIC16 (10 µM for 10 min at 37°C). When used as targets for CTL, they were subsequently surface TNP-modified by incubation with 1 mM trinitrobenzene sulfonate in PBS, pH 7.4, for 15 min at 37°C followed by washing.
The CTL hybridoma d11S was preactivated to express FasL by incubation for 3 h with PMA (5 ng/ml) and ionomycin (3 µg/ml) before the cytotoxicity assay. CTL were generated from primary in vitro mixed lymphocyte cultures in which spleen cells from BALB/c (H-2d) mice were mixed with irradiated (300 Gy) splenocytes from C57BL/6 (H-2b) mice at a ratio of 2.5:1 and incubated for 5 days in complete medium at 37°C. Further culture was conducted at 5 x 105 cells/ml in complete medium with 10 U/ml of rIL-2. Effector cells were harvested by centrifugation over lympholyte M (Accurate Chemical and Scientific Corp., Westbury, NJ). NK effector cells were obtained from human PBMC centrifuged over Ficoll-Hypaque (Sigma) and used directly or activated by overnight culture at 5 x 106/ml in complete medium with 20 U/ml IL-2.
Assays of target damage
All experiments assessing the CTL granule exocytosis pathway
used primary cultures of allo-CTL redirected to TNP target cells with
100 ng/ml 
CD3x--TNP heteroconjugate as previously described
(17). In these incubations the Fas cytotoxicity pathway was blocked by
addition of 10 µg/ml of IgG anti-Fas mAb DX2, which completely
blocked Jurkat lysis by d11S CTL. Incubations were conducted in
flat-bottom microtiter wells containing 104 target cells,
which, when indicated, were pretreated with peptide-FMK caspase
inhibitors for 1 h before addition of effector cells. The
indicated number of effector cells was added and centrifuged at 300
rpm. The plates were then placed in a 37°C CO2 incubator
at time zero. At the indicated times wells were harvested by pipetting,
and target damage was assessed by flow cytometry after pooling an
appropriate number of wells.
To assess nuclear morphology and formation of membrane blebs, cells were stained with 5 µg/ml Hoechst 33342, and DiIC16-positive cells were scored for apoptotic nuclear morphology or the presence of blebs by fluorescence microscopy. PS exposure was assayed by flow cytometry after staining with 200 ng/ml annexin V-FITC in HEPES-buffered saline containing 2.5 mM CaCl2 for 15 min in the dark and suspending in this buffer containing 10 µg/ml PI. The DiIC16-positive, PI-negative subset of cells was analyzed for fluorescein fluorescence intensity. Mitochondrial potential was estimated using DiOC6 (3, 18) by incubation of the resuspended cell pellets with 40 nM DiOC6 (3) in 0.5 ml of PBS for 20 min in the dark at 37°C. After centrifugation the cells were resuspended in 500 µl of PBS containing 10 µg/ml PI, and the DiIC16-positive, PI-negative subset of cells was immediately analyzed by flow cytometry.
Plasma membrane recycling was measured by incubation of the cell suspension with 5 mM FM1–43 for 10 min and washing three times in complete medium before analysis on the flow cytometer. Because of the fluorescence properties of FM1–43, it was not possible to identify target cells with DiIC16, and in these experiments target cells were selectively analyzed using a scatter gate (two-dimensional forward and side scatter) defined by running effectors and targets alone. This approach limited these experiments to modest E:T cell ratios.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human PBL were tested for their ability to lyse and induce target
nuclear damage in the classic NK target K562. As shown in Figure 1
A, a 4-h incubation of normal
PBL with K562 target cells at an E:T cell ratio of 20 resulted in both
lysis and apoptotic nuclear damage of these targets, confirming
previous results with these apoptosis-resistant BCR-overexpressing
target cells (19). The dependence of these cytotoxic effects on
caspases was tested using the cell-permeable peptide-based caspase
inhibitors ZVAD-FMK and BD-FMK, which were previously shown to block a
variety of death readouts in lymphocytes treated with various
apoptogenic agents (20). These caspase inhibitors caused a
dose-dependent inhibition of K562 apoptotic nuclear damage, while the
control reagent ZFA-FMK slightly enhanced such damage. In contrast,
none of these peptide-FMK reagents had a significant effect on target
lysis. These results are strikingly parallel to those previously
obtained with mouse CTL using the granule exocytosis effector
pathway (10).
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B. The
considerably more potent cytotoxic activity of these activated NK cells
showed a similar pattern of blocking by caspase inhibitors as fresh NK
cells, i.e., potent blocking of nuclear damage by ZVAD-FMK, slightly
weaker blocking by BD-FMK, and no effect of ZFA-FMK, while there was no
effect on target lysis. In other experiments we further confirmed these
findings using adherent activated mouse spleen NK cells with mouse YAC
tumor targets (data not shown). The CTL FasL/Fas effector pathway induces a caspase-dependent target cell PS exposure before lysis
Because our objective was to examine the nonnuclear apoptotic
events induced in target cells by cytotoxic lymphocytes before lysis,
we have used flow cytometry to identify PI-negative target cells
prelabeled with a lipid probe and followed a third marker of apoptotic
damage. We have separately tested the granule exocytosis and FasL/Fas
effector pathways, since these together account for rapid in vitro
CTL-mediated target cell death (1). We first examined the surface
exposure of PS by binding of FITC-annexin V. Figure 2A shows that the FasL-bearing
effector cell d11S (21) mediates a rapid PS exposure on PI-negative
Jurkat target cells at modest E:T cell ratios. Figure 2B
indicates that this PS exposure as well as the induction of apoptotic
nuclear morphology are specifically blocked by peptide-FMK caspase
inhibitors. This result is predicted by a pathway involving Fas-induced
cross-linking of caspase 8 or 10 via FADD and was expected in
light of previous results showing that PS exposure induced by
anti-Fas Ab is caspase dependent (22, 23).
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Figure 3A shows that
primary in vitro-generated CTL, in the presence of IgG anti-Fas to
block the Fas killing pathway, mediate a rapid prelytic PS exposure in
Jurkat target cells. In Figure 3B we tested the ability of
ZVAD-FMK and BD-FMK to block this PS exposure and observed a dramatic
contrast to the results presented in Figure 2B. In this
case, peptide-FMK caspase inhibitors efficiently blocked apoptotic
nuclear morphology (confirming our previous results (10)), but had no
effect on PS exposure.
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. In this case
PS exposure was extremely rapid, with >35% of target cells showing
increased annexin V staining within 20 min at an E:T cell ratio of 10.
However, this PS exposure was not detectably inhibited by peptide-FMK
caspase inhibitors, although these inhibitors blocked apoptotic nuclear
damage in the same experiment (Fig. 4B).
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Dying cells have been reported to undergo a rapid loss of
mitochondrial potential , as measured by DiOC6
fluorescence (24). To determine whether CTL targets undergo a rapid
prelytic loss of mitochondrial potential, we used a similar analysis by
flow cytometry. Figure 5A
shows that the FasL-bearing d11S cells induce a loss of mitochondrial
that is detectable within 1 h, and Figure 6A shows that CTL using the
granule exocytosis pathway show a generally similar activity. The
ability of peptide-FMK caspase inhibitors to block this mitochondrial
damage was then tested. As shown in Figure 5B, the prelytic
mitochondrial loss induced by the FasL-bearing d11S CTL was
dramatically and specifically blocked by these inhibitors along with
nuclear damage. In contrast, loss of via the CTL granule exocytosis
pathway was not detectably inhibited by the caspase inhibitors,
although in the same cells apoptotic nuclear damage was effectively and
specifically inhibited (Fig. 6B).
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Membrane blebbing has long been recognized as an early sign of
CTL-induced target cell injury (25, 26) and is part of the classical
syndrome of apoptotic death (27). We have found that the
DiIC16 lipid probe used in the above experiments to
distinguish target cells from CTL affords a reliable means of
visualizing target plasma membrane blebs in the fluorescent microscope.
Using Jurkat target cells and the CTL effector systems described
above, we observed a rapid induction of blebs by both the granule
exocytosis and FasL/Fas pathways, with 40 to 50% of the target cells
showing clear membrane blebs by 90 min at modest E:T cell ratios (Fig. 7). When peptide-FMK caspase inhibitors
were tested for the ability to block this bleb formation, they were
found to specifically block that induced by d11S, but not primary CTL
using the granule exocytosis pathway.
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CTL have been reported to rapidly induce an increase in staining
of target cells after a pulse exposure to the lipid probe FM1–43 (28).
Such increased FM1–43 staining is considered a measure of endocytic
membrane internalization and is found at neuronal synapses after
transmitter release (29, 30). Figure 8
shows the rapid increase in FM1–43 staining in Jurkat target cells
induced by the CTL granule exocytosis pathway. Figure 8A
shows that 90 min after mixing CTL with target cells under conditions
of TCR engagement there was a clear subpopulation of target cells with
increased FM1–43 fluorescence. This increase was inhibited by the
caspase inhibitors BD-FMK and ZVAD-FMK, but not by the control compound
ZFA-FMK (Fig. 8B). Thus, increased FM1–43 staining is one
measure of nonnuclear damage induced by CTL granule exocytosis that is
blocked by peptide-FMK caspase inhibitors and thus appears to be
similar to nuclear damage in its sensitivity to caspase inhibitors.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The above examples of target damage induced by CTL are not surprising
in light of previous demonstrations that CTL activate caspases, since
caspase inhibitors block all measurable apoptotic damage by a wide
range of apoptotic triggers. However, the most interesting aspect of
the experiments described here is that three of the four measurements
of target damage induced by the granule exocytosis pathway were not
blocked by caspase inhibitors that completely blocked nuclear damage in
the same experiment. This means that the these nonnuclear events do not
require the granzyme-induced caspase activation that leads to nuclear
damage, part of which is probably attributable to the recently
described caspase-activated DNase (31). Since these nonnuclear changes
are typically associated with apoptotic death, the results strongly
suggest that granzymes directly trigger a nonnuclear molecular death
pathway that leads to mitochondrial depolarization, membrane PS
externalization and blebbing, and lysis, as shown in Figure 9. The molecular nature of the
postcaspase cell destruction pathway has not been defined in any
system. While it is sometimes assumed that caspase-induced nuclear
damage leads to cell death, this is clearly not the case for CTL, since
enucleated target cells are rapidly lysed by both pathways (32). We
find it plausible that granzymes and caspases have common substrates
that initiate this downstream death pathway, as shown in Figure 9.
While a number of nuclear substrates of caspases have been identified,
the critical substrates that initiate the pathway leading to lysis
remain unidentified.
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One limitation of our experiments is the use of caspase inhibitors whose ability to block intracellular caspase activation is incompletely defined. For that reason, we previously showed (6, 10) that CTL granule exocytosis-induced target lysis was not affected by baculovirus p35, which inhibits caspases 1 to 4 at a 1:1 molar ratio (34). We have also previously shown that the peptide FMK inhibitors used in this paper block the DEVDase activity characteristic of caspases 3 and 7 in extracts of apoptotic Jurkat cells (20). However, it is not clear how to compare the activities of such caspases in extracts with those in intact cells, and it is not established which of the 10 known caspases are directly inactivated by the reagent conditions used. Thus, it is quite possible that these peptide-FMK inhibitors block the Fas death pathway by selectively reacting with caspases 8 and 10, which may be the targets of granzyme B (12). One can postulate that the failure of peptide-FMK caspase inhibitors to block lysis and nonnuclear damage by the granule exocytosis pathway is due to their failure to block the relevant granzyme B-activated caspases. However, such an explanation does not address why these inhibitors so effectively block nuclear damage in this pathway, and one would thus need to postulate that nuclear damage requires caspases sensitive to these inhibitors but that lysis and other nonnuclear damage use caspases that are resistant. Granule exocytosis-triggered lysis of T lymphoid cells appears unique among apoptotic agents in its resistance to inhibition by peptide-FMKs, as we have shown that peptide-FMK caspase inhibitors effectively and specifically block lysis of Jurkat target cells induced by anti-Fas, TNF, staurosporine, vinblastine, and several DNA-damaging chemotherapeutic agents (10, 35). In this respect Jurkat cells reflect the apoptotic lysis of most T lymphoid cells induced by a wide variety of agents (20). Thus, while we cannot formally rule out a role for peptide-FMK-resistant caspases as described above, our experiments show that it is very likely that CTL-injected granzymes act by directly triggering a downstream apoptotic death pathway without the involvement of caspases.
While unusual, there are several other reported examples of apoptotic cell death which are resistant to caspase inhibitors, and it will be of interest to determine whether these deaths share molecular steps with death induced by lymphocyte granule exocytosis. In fibroblasts, ZVAD-FMK and BD-FMK retard, but do not, prevent apoptotic nuclear damage and cell death induced by several agents (36), and defects in the ubiquitin-activating enzyme E1 lead to an apparently caspase-independent death (37). Some examples of apoptotic death resistant to ZVAD-FMK have been reported (38, 39), but we have found cases where the analogous caspase inhibitor BD-FMK specifically blocks death (20).
Externalization of PS is a functionally important manifestation of apoptotic death, since this triggers one of the recognition systems used by macrophages to phagocytose apoptotic cells (40). This lipid is normally restricted to the inner membrane leaflet but becomes detectable externally before cytolysis during apoptotic death (41). For the rapid target death induced by cytotoxic lymphocytes it has not been previously shown that PS externalization occurs before lysis, and our findings suggest that cells killed by CTL in vivo are likely to be rapidly phagocytosed.
The molecular pathways leading to PS externalization during apoptosis are not well understood. In Jurkat cells treated with anti-Fas this process requires active caspases and calcium in the medium (42, 43). A plausible model for this would be that caspase activation results in an elevation of intracellular calcium (44), thus activating the lipid scramblase that mediates transbilayer phospholipid "flip-flop" (45). For the granule exocytosis death pathway, it is likely that perforin pores also allow leakage of external calcium across the target membrane, although this may be temporary due to membrane repair processes. Thus, additional granule components such as granzymes may also be required to trigger PS exposure, and its calcium dependence is not readily tested, since calcium is also required for degranulation. It is clearly important to design future experiments to probe this issue and to establish whether target lysis is closely linked to PS exposure.
The prelytic collapse of mitochondrial potential is associated with
the mitochondrial permeability transition and may occur in both
apoptotic and nonapoptotic death (46). An associated release of a
mitochondrial apoptosis-inducing factor has been proposed to be a
critical step in apoptotic signaling (47). As shown for the
CTL-mediated Fas death pathway in Figure 5B, ZVAD-FMK blocks
the anti-Fas-induced loss of mitochondrial electron transport in
Jurkat cells (48), the UV B-induced loss of mitochondrial in CEM
cells (49), and some mitochondrial damage in apoptotic thymocytes (46).
For death induced by the granule exocytosis pathway, induction of
mitochondrial damage is one candidate functional role for granzymes.
Granzymes could be envisioned to trigger the release of mitochondrial
cytochrome c, thus activating procaspase 9 via Apaf-1 (50, 51), or triggering the permeability transition and release of
apoptosis-inducing factor (47). However, both of these are proposed to
trigger death via activation of downstream caspases, making such models
unattractive in light of the present evidence for caspase independence.
The increase in FM1–43 pulse staining induced by the CTL granule
exocytosis pathway (Fig. 8) confirms a previous report that both CTL
effector pathways rapidly induce an enhancement in plasma membrane
endocytosis into internal vesicles revealed by this technique (28).
This fluorescent lipid probe rapidly and reversibly partitions into the
outer bilayer leaflet from the medium, so that postpulse washing
removes it from the exposed plasma membrane but not from membrane that
underwent endocytosis during the pulse. The increased rate of plasma
membrane uptake is a property of other apoptotic death systems as well,
including thymocytes treated with dexamethasone and etoposide in vitro
(28) (E. K. Haddad, unpublished observations). While it seemed
plausible to assume that such enhanced endocytosis was part of the
apoptotic membrane dysregulation also resulting in blebbing and PS
exposure, Figure 8 shows that for CTL granule exocytosis-induced target
damage, this enhanced endocytosis is part of a caspase-dependent
pathway and hence distinct from the other types of membrane damage.
These results also show that nuclear damage is not the only
caspase-dependent damage induced by the granule exocytosis pathway.
Further molecular definition of the postcaspase cell destruction pathway is clearly of interest. The present results are helpful in that regard because they suggest that identification of critical protein substrates cleaved by both granzymes and caspases may lead to identification of critical players triggering this pathway.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Pierre Henkart, Building 10, Room 4B17, National Institutes of Health, Bethesda, MD 20892-1360.
3 Abbreviations used in this paper: FasL, Fas ligand; PS, phosphatidylserine; ZVAD-FMK, N-carbobenzoxy-valyl-alanyl-aspartic acid (O-methyl)fluoromethyl ketone; BD-FMK, N-tert-butoxy-carbonyl-aspartic acid (O-methyl)fluoromethyl ketone; ZFA-FMK, N-carbobenzoxy-phenylalanyl-alanine fluoromethyl ketone; FM1–43, N-(3-triethylammoniumpropyl)-4-[4-(dibutylamino)styryl]pyridinium dibromide; DiIC16, 1,1'-dihexadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate; DiOC6, 3,3'-dihexyloxacarbocyanine iodide; TNP, trinitrophenyl; PI, propidium iodide.
Received for publication March 23, 1998. Accepted for publication May 19, 1998.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A. Shcherbina and E. Remold-O'Donnell Role of Caspase in a Subset of Human Platelet Activation Responses Blood, June 15, 1999; 93(12): 4222 - 4231. [Abstract] [Full Text] [PDF]
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K. A. Browne, R. W. Johnstone, D. A. Jans, and J. A. Trapani Filamin (280-kDa Actin-binding Protein) Is a Caspase Substrate and Is Also Cleaved Directly by the Cytotoxic T Lymphocyte Protease Granzyme B during Apoptosis J. Biol. Chem., December 8, 2000; 275(50): 39262 - 39266. [Abstract] [Full Text] [PDF]
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D. Zhang, M. S. Pasternack, P. J. Beresford, L. Wagner, A. H. Greenberg, and J. Lieberman Induction of Rapid Histone Degradation by the Cytotoxic T Lymphocyte Protease Granzyme A J. Biol. Chem., January 26, 2001; 276(5): 3683 - 3690. [Abstract] [Full Text] [PDF]
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D. Zhang, P. J. Beresford, A. H. Greenberg, and J. Lieberman Granzymes A and B directly cleave lamins and disrupt the nuclear lamina during granule-mediated cytolysis PNAS, May 8, 2001; 98(10): 5746 - 5751. [Abstract] [Full Text] [PDF]
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