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*
OncoImmunin, Inc., Gaithersburg, MD 20877; and
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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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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14 intracellular cysteine proteases
related by sequence homology to IL-1
-processing enzyme. Caspases are
characterized by a pronounced proteolytic cleavage preference for
aspartic acid residues in the P1 position, with
preferences for the P4 and to some extent
P3 and P2 positions
differing among the different family members (2). Much
current effort is focused on the mechanisms of activation of these
proteases, which are expressed intracellularly as proenzymes requiring
enzymatic processing to become active (3). While it is
clear that active caspases can often process other procaspases in a
cascade, the initiating events appear to be recruitment of procaspase 9
or 8 into complexes that promote autoprocessing. Affinity-labeling
studies have shown that caspases 3, 6, and 7 often dominate caspase
activity in apoptotic cells (4). However, since a host of
regulatory elements operate on the formation of caspase initiation
complexes and also modulate activity of individual proteases, the
caspase cascade itself may differ according to apoptotic stimulus and
cell type. Analysis of the apoptotic caspase cascade is generally conducted by using mAb against individual caspases with Western blots to monitor proteolytic processing events accompanying activation as assessed on a cell population basis. While such measurements monitor processing of individual caspases, their drawbacks are that processing may not reflect proteolytic activation, and information on cellular heterogeneity and intracellular compartmentalization is lost during the preparation of extracts. We have developed an alternative and complementary approach to monitoring caspase cascades that utilizes a series of cell-permeable fluorogenic substrates capable of monitoring caspase activities in intact apoptotic cells, and we have previously used this approach with flow cytometry and confocal microscopy to analyze caspases in thymocytes undergoing apoptosis triggered by corticosteroid and anti-Fas (5).
In extending our studies to cells other than thymocytes, the Fas apoptotic pathway was attractive because it is well studied, and variations in caspase cascades have been reported in different cell types (6). In the case of mature T cells, TCR-induced Fas ligand expression can lead to suicidal or fratricidal death via the Fas pathway, and this activation-induced cell death may limit immune responses in the face of high Ag loads (7). After receptor aggregation and recruitment of Fas-associated death domain protein and procaspase 8 to a signaling complex (death-inducing signaling complex (DISC)),3 caspase 8 becomes activated. This can directly lead to processing and activation of effector caspases 3, 7, and 6. Alternatively, active caspase 8 can cleave Bid, yielding a truncated species that inserts into the outer mitochondrial membrane and facilitates cytochrome c release and Apaf-1-mediated activation of caspase 9 (8). When we sought to test whether our caspase substrates were capable of distinguishing between these two caspase cascades, we observed strikingly early caspase activity in surface membrane blebs. We describe these novel findings in the present communication.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RPMI medium and FCS were purchased from HyClone (Logan, UT), and AIM-V medium from Life Technologies (Gaithersburg, MD). Mouse IgM anti-human Fas (CD95) Ab (catalogue 2387) was from Coulter (Miami, FL); IL-2 from Cetus/Chiron (Emeryville, CA); IL-7, ZVAD(OMe)-FMK, and Hoechst 33342 from Calbiochem (San Diego, CA); 3,3'-dihexyloxacarbocyanine iodide (DiOC6), propidium iodide (PI), and Sytox Green from Molecular Probes (Eugene, OR); and PI/RNase staining buffer from Phoenix Flow Systems (San Diego, CA). Solvents such as HPLC grade water, dichloromethane, methanol, and acetonitrile were from VWR Scientific Products (South Plainfield, NJ). Reverse-phase HPLC equipment and columns were from Waters (Milford, MA) and SynChrom (Lafayette, IN).
The human CTL line used in this study was originally derived from a human melanoma tumor-infiltrating lymphocyte in AIM-V medium in the presence of 6000 IU IL-2 (9). The cells are CD8+ and can be grown indefinitely in the continuous presence of IL-2 in either AIM-V or RPMI medium containing 10% FCS. Jurkat and SKW 6.4 cell lines were purchased from the American Type Culture Collection (Manassas, VA) and grown in RPMI plus 10% FCS. Apoptosis was induced by addition of anti-Fas at concentrations ranging from 200 to 400 ng/ml.
Caspase substrates
The reagents and methods used for peptide synthesis and
derivatization have been described in detail previously
(10). Briefly, peptides were synthesized using an
automated peptide synthesizer and by manual solid-phase methodology and
subsequently purified by reverse-phase HPLC. Peptides were subjected to
mass spectrometric analysis at PeptidoGenetic (Livermore, CA) to
determine the molecular mass and confirm peptide structure and
composition. Each purified peptide was derivatized with the appropriate
fluorophore, as previously described (5). Substrates were
purified into single components of homodoubly derivatized peptides by
reverse-phase HPLC and further characterized by absorption and
fluorescence spectroscopy. For a list of caspase substrates and their
cleavage sequences, see Table I
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Flow cytometry. Incubation of cells with fluorogenic substrates was conducted each hour starting at the time of anti-Fas addition, as described previously (5). Additionally, DiOC6 (final concentration of 0.5 µM) was added to a separate tube containing cells, incubated for 15 min at 37°C, washed, anti-Fas added, and the CTL analyzed by flow cytometry in parallel with the substrate-exposed cells. All measurements were made with a Coulter (EPIC-XL) flow cytometer using EXPO 2 analysis software. Ten thousand PI-negative cellular events were collected for each file using FL1 vs FL3 dot plots to establish a PI-negative polygonal gate. Throughout the entire time course of experiments, the determined PI-positive population of any sample was never greater than 15% of the total.
Microscopy
Cells were placed in medium containing fluorogenic substrates at 2 µM each with or without Sytox Green at 10 nM and anti-Fas in a temperature-controlled imaging chamber from Bioptechs (Butler, PA) and viewed on a Zeiss LSM410 laser-scanning confocal microscope system using a x63, 1.4N.A. objective at 37°C with an objective heater. Samples were excited using the 488-nm and/or 568-nm Krypton/Argon laser lines and/or the 633-nm line of a red He/Ne laser. For time course studies, fluorescence and differential interference contrast (DIC) images were acquired every minute. Fluorescence images were acquired as single optical sections of 1 µm in thickness, and brightness/contrast settings were adjusted such that the fluorescence signals of cells before substrate cleavage were at or slightly below background. As substrates were cleaved in apoptotic cells, cellular fluorescence rose above the fluorescence of the bulk solution in the same optical plane. Changes in cell size and fluorescence were analyzed using Imaris software from Bitplane AG (Zurich, Switzerland). For analyzing the effects of ZVAD-FMK on blebbing, cultures were viewed with an Olympus IX70 microscope by phase and, after centrifuging and washing, fluorescence microscopy to localize IETDase activity.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our previous study using cell-permeable fluorogenic substrates to
examine caspase cascades was conducted in thymocytes treated with a
corticosteroid or anti-Fas Ab. We have subsequently used this
approach to examine caspase activities in mature effector T cells,
i.e., cytotoxic T cells, after treatment with anti-Fas. Fas
cross-linking in these CTL induced an apoptotic mitochondrial
depolarization detectable within 1 h, as shown by flow cytometry
using the potential sensitive dye DiOC6 (Fig. 1A). The mitochondrial
potential continued to decrease further until 5 h posttreatment.
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A). With increasing time, a discrete second peak,
indicative of substrate cleavage, appeared for each substrate. Looking
at the 2- to 4-h time points, a definite order of caspase activation
can be inferred. When the percentages of caspase+
cells were plotted as a function of induction time (Fig. 1B), time of activation of the five caspase
activities fell into two groups: YVHDase, LEHDase, and VEIDase in an
early group, and DEVDase and IETDase in a later group that lagged the
first by about 1 h. The late appearance of the IETDase activity is
noteworthy, as this activity was the earliest measured during
anti-Fas-induced apoptosis in thymocytes, and is predicted to be
autoactivated in the DISC complex comprised of procaspase 8, Fas, and
Fas-associated death domain protein.
The fluorogenic caspase substrates can be used for microscopy to
complement the quantitative data from flow cytometry. Fig. 1C shows a confocal fluorescence micrograph of CTL 2 h
after addition of anti-Fas. The field is representative of many
examined fields, which included a total of several hundred cells.
Individual cells showing only green fluorescence from
DiOC6 have maintained a high mitochondrial
potential and are not clearly apoptotic. The brightest green-staining
cells have no detectable red staining due to cleavage of the YVHD
substrate (one of the early group). Cells with diminished mitochondrial
potential (green stain) are apoptotic and display red cytoplasmic
fluorescence, indicative of YVHDase activity. Thus, the intracellular
caspase activity fluorescence together with the diminishing green
reflects apoptosis progression in individual cells as analyzed by
microscopy.
Subcellular localization of apoptotic activities
To gain insight into the late appearance of the IETDase activity
by flow cytometry, we examined anti-Fas-treated CTL loaded with a
red IETDase substrate in the presence of Sytox green, an ionic
DNA-intercalating dye functionally analogous to PI. Fig. 2 shows a series of confocal images of
one CTL taken 1 min apart, starting at the beginning of the third hour
after anti-Fas addition. Surface membrane blebs, containing IETDase
activity, are visible and are being continuously generated by this
cell, before the loss of its plasma membrane integrity, which is
reflected in the Sytox green staining in the final frames.
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A shows CTL loaded with
a combination of the IETDase substrate (red) and the VEIDase (green).
These were chosen to compare early and late activities observed by flow
cytometry (Fig. 1B). The field selected, representative of
many fields studied, contains cells at various stages of apoptosis,
ranging from early (very round cells) to late (fragmented cells). The
thickness of the 1-µm optical section may influence the appearance in
an individual cell; however, our observations are consistent with
red-only cells containing IETDase activity near the plasma membrane or
in bleb- containing cells at various stages of apoptosis ranging
from early (very round cells) to late (fragmented cells). The smooth
round cells show considerably less IETDase than those that are more
granular and less spherical, as well as many
IETDase+ blebs. Additionally, one cell is
VEIDase+ with cytoplasmic IETDase developing.
Both the red blebs and the remaining whole cells eventually became
double stained (yellow) due to VEIDase activation subsequent to IETDase
activation.
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, B and C) with the same
substrates, but with the fluorophores reversed compared with those of
Fig. 3A. Fig. 3B shows a single cell with
IETDase-positive blebs and modest VEIDase activity in its cytoplasm.
Fig. 3C shows an example from later times in which all the
cells have become VEIDase+. At the later time,
blebs positive for both VEIDase and IETDase can be seen. At
such later times, blebs were found to be positive for YVHDase and
DEVDase as well. Regardless of the labeling, we have not observed significant amounts of IETDase signal alone in the cytoplasm of CTL after Fas cross-linking. Early IETDase activity is produced mainly at the surface or within the blebs. Moreover, the blebs become yellow due to activation of VEIDase activity. The majority of the early IETDase activity is present in blebs that were either detached from cells or that would likely become detached by the shearing forces expected in flow cytometry. Lack of detection of IETDase activity in some blebs may be due to the level and thinness of the optical section. Blebbing, specifically formation of IETDase-containing blebs, was found to be dependent on caspase activation, as the presence of 50 µM ZVAD(OMe)-FMK in the inducing medium abolished this process (data not shown).
Apoptotic CTL deprived of survival factor show cytoplasmic IETDase activity
The CTL line used in these experiments was maintained in IL-2, and
had become IL-2 dependent, as seen by a slow apoptotic
deathover several days after IL-2 withdrawal. When such
apoptotic cells were loaded with the IETDase and DEVDase substrates,
flow cytometry showed an early increase in cytoplasmic IETDase,
followed by an increase in DEVDase (data not shown). Fig. 4 shows a time at which all the
DEVDase+ cells are also
IETDase+ and other IETDase+
cells are DEVDase-. Unlike the case of
anti-Fas-induced death, there was a notable absence of plasma
membrane blebs in these cultures.
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To determine the generality of the above observations, we examined
activation of the five caspases in other lymphoid cell lines. In
particular, the Burkitt’s lymphoma SKW6.4 line was examined
because caspase 8 activity has been shown to clearly precede other
caspases after anti-Fas treatment. In these cells, IETDase activity
(red) appears in the cytoplasm before DEVDase activity (green). Fig. 5 shows a relatively early stage of this
death in which many cells are
IETDase+DEVDase-, with one
IETDase-DEVDase+ cell and
no double-positive cells. Some cells display plasma membrane blebs that
in this case are IETDase negative. When Jurkat cells were examined
after anti-Fas treatment, caspase activities were found to be more
similar to those expressed in the CTLs; however, resolution in terms of
time as well as morphology was not as well defined (data not
shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The histograms in Fig. 1A show that cross-linking of Fas on
CTL gives rise to an ordered increase in caspase activities.
Nonapoptotic CTL show largely homogeneous histograms representing
background fluorescence, which over the hours immediately following
anti-Fas treatment gave rise to a second relatively homogeneous
population of cells with increased fluorescence due to caspase
activation. Fig. 1B shows that the measured caspase
activities cluster into two groups based on their time of increase
after Fas cross-linking: VEIDase, LEHDase, and YVHDase in one, and a
second slower group containing DEVDase and IETDase. The relatively late
activation of IETDase was quite surprising in view of the
well-established model that Fas cross-linking leads to recruitment of
pro-caspase 8 into a DISC, which promotes autoactivation, initiating
the caspase cascade (6). Our previous studies
(5) with anti-Fas-treated thymocytes showed that
IETDase activation occurred before other caspases, as expected from
this model. Thus, the thymocyte results also make it unlikely that the
late IETDase activation is due to a lower detection sensitivity of this
substrate to caspase 8.
When anti-Fas-treated CTL loaded with the IETD substrate were
examined in the confocal microscope, we were surprised to find that
activity was largely confined to membrane blebs forming within the
first 2 h (Figs. 2 and 3). This IETDase activity preceded the
appearance of YVHDase, VEIDase, and DEVDase activities, which were
cytoplasmic. Membrane blebbing has long been associated with apoptosis,
but the molecular basis for this phenomenon is not completely clear.
There appear to be two types of apoptotic blebbing. One type occurs in
lymphoid cells and hepatocytes after Fas cross-linking and is blocked
by caspase inhibitors. In this case (11, 12),
caspase 3-mediated fodrin cleavage has been proposed as a mechanism
(13, 14). A second type of apoptotic blebbing, seen in
fibroblasts, PC12 cells, and COS cells, is resistant to caspase
inhibitors and is regulated by myosin L chain phosphorylation
(15). Fas-induced apoptosis in CTL would be expected to be
in the first category, and indeed the early blebbing induced by
anti-Fas is completely blocked by ZVAD-FMK. Thus, local caspase
8-mediated caspase 3 activation may be responsible for bleb formation,
but is not detectable at this early time by the DEVDase substrate.
Because the processing associated with caspase 8 activation may
liberate this enzyme from the DISC, it might have been presumed
that active caspase 8 would have diffused away from the membrane
before bleb formation. However, since this was not observed, it seems
likely that active caspase 8 is still associated with the plasma
membrane, either because the second processing cleavage liberating the
p18 chain from the propeptide has not occurred, or because the p18/p10
caspase dimer is locally bound.
To account for differences among individual cells in the apoptotic response to Fas ligation, it has been proposed that cells die by one of two signaling pathways (16). In type 1 cells, Fas receptor aggregation recruits signaling molecules to the plasma membrane via formation of the DISC, which promotes caspase 8 autoactivation with subsequent activation of downstream effector caspases. In type II cells, the model proposes that DISC formation is strongly reduced relative to type I cells, so that the low amounts of active caspase 8 are not sufficient to initiate a general caspase cascade. However, caspase 8 cleaves Bid, which can then damage mitochondria, inducing cytochrome c release and caspase 9 activation via Apaf-1. Both pathways are proposed to contribute to different extents in various cell types.
To relate our observations with CTL to this two-pathway model, we
examined activation of the five caspases in other cell lines with an
emphasis on SKW6.4 (Burkitt’s lymphoma), a prototype type I cell, and
Jurkat, a prototype type II cell (16). Our results with
SKW6.4 and Jurkat cells were not inconsistent with this model. For
example, flow cytometry data for the Jurkat cells were similar to those
reported in this work for CTL in that the IETDase activity did not
precede other caspases in the majority of cells. In contrast, flow
cytometry of anti-Fas-treated SKW6.4 cells showed that IETDase
activity did precede other caspases, and microscopy revealed
insignificant bleb formation in these cells relative to the CTL
(Fig. 4).
Our observations of caspase 8 activation in blebs may shed light on the proposed two-pathway model. If caspase 8 has been activated at the DISC and lost in blebs from apoptotic type II cells, biochemical analysis of the remaining cells will fail to detect this activity because the blebs are not monitored. It is thus possible that some of the experimental differences used to distinguish between type I and type II cells are attributable to differences in early bleb formation and removal of active enzyme from the cells studied.
To further evaluate the significance of caspase activities in the
physiology of whole cells, we compared cleavage of the same five
substrates in CTL deprived of IL-2. In this case, there was minimal
blebbing, and IETDase was localized inside the cells (Fig. 5). Thus,
apoptosis in the IL-2-deprived CTL provides a striking contrast to that
induced by anti-Fas, showing that IETDase activity originates from
different cellular components with different apoptotic triggers.
Thus, simultaneous visualization of multiple caspase activities has provided insight into the mechanism of caspase activation early in the apoptotic protease cascade. The combination of flow cytometry and confocal microscopy data presented in this study shows compartmentalization of IETDase activity in blebs, unanticipated from previous extensive biochemical studies on Fas-induced apoptosis. This localization appears to be a reasonable consequence of IETDase activation in DISCs after Fas cross-linking. Consideration of cellular compartmentalization of molecular activities is essential for meaningful conclusions about the caspase cascade and apoptotic signaling.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Pierre A. Henkart, National Institutes of Health, Building 10, Room 4B36, Bethesda, MD 20892-1360. E-mail address: ph8j{at}nih.gov
3 Abbreviations used in this paper: DISC, death-inducing signaling complex; DIC, differential interference contrast; DiOC6, 3,3'-dihexyloxacarbocyanine iodide; PI, propidium iodide.
Received for publication June 19, 2001. Accepted for publication August 24, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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