1 Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de
Chile, Santiago, Chile
2 Institute of Biochemistry, University of Lausanne, Switzerland
* Author for correspondence (e-mail:aquest{at}machi.med.uchile.cl)
Accepted 4 September 2002
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Summary |
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Key words: Lymphoid cells, Fas, Apoptosis, Necrosis, Caspases, Ceramide
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Introduction |
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FasL binding stabilizes the trimeric form of the Fas receptor, thereby
allowing recruitment of the Fas-associated death domain (DD)-containing
protein (FADD). FADD then binds to and activates caspase-8, an initiator
caspase, which in turn activates downstream effector caspases, including
caspase-3 (Martins and Earnshaw,
1997; Nagata and Golstein,
1995
; Suda et al.,
1993
). As a consequence, many cellular proteins are degraded,
leading to cell death.
Depending on the cell type and the stimulus, a cell may die either by
apoptosis or necrosis. Apoptosis is characterized by chromatin condensation,
internucleosomal degradation of the DNA, cell shrinkage and disassembly into
membrane-enclosed vesicles as a consequence of caspase activation
(Rathmell and Thompson, 1999).
Apoptotic and necrotic cells are both recognized by phagocytes, but only
apoptotic cells are eliminated without release of cytosolic components to the
environment, thereby preventing an inflammatory response
(Fadok et al., 2000
;
Sauter et al., 2000
).
Necrosis, on the other hand, is characterized by swelling of the cells and
organelles, resulting ultimately in disruption of the cell membrane and cell
lysis (Majno and Joris,
1995
).
Initially, FasL was only thought to trigger cell death by apoptosis.
Recently, however, inhibition of Fas-induced apoptosis in L929 fibrosarcoma
cells by a caspase inhibitor lead to necrosis mediated by oxygen radicals
(Vercammen et al., 1998a;
Vercammen et al., 1998b
).
Also, primary activated T cells can be efficiently killed by FasL, TNF-
and TRAIL in the absence of active caspases
(Holler et al., 2000
). These
results suggested that Fas, like TNFR-1
(Laster et al., 1988
),
triggers apoptotic or necrotic death.
Treatment with FasL, TNF- or IL-1ß leads to formation of
ceramide, in addition to caspase-3 activation
(Garcia-Ruiz et al., 1997
;
Gudz et al., 1997
). Ceramide
reportedly modulates the activity of a large number of proteins
(Heinrich et al., 1999
;
Venkataraman and Futerman,
2000
) and, in doing so, may promote apoptosis. In addition,
ceramide directly modulates mitochondria function, for instance by inhibiting
the mitochondrial respiratory complex III
(Garcia-Ruiz et al., 1997
;
Gudz et al., 1997
;
Quillet-Mary et al., 1997
).
Sphingomyelin hydrolysis by either neutral (nSMase) or acidic
sphingomyelinases (aSMase) is generally implicated in ceramide production
(Hannun et al., 1996
;
Kolesnick and Kronke,
1998
).
Treatment of lymphoid cells with FasL induces elevation of intracellular
ceramide levels. However, data are conflicting with regard to the kinetics of
the ceramide response and the SMases involved downstream of Fas. Some authors
have found a rapid transient response within minutes to an hour that depends
on initiator caspases, like caspase-8, and has been attributed to activation
of an aSMase (Cifone et al.,
1995; Genestier et al.,
1998
). Alternatively, others have reported that apoptosis of
lymphoid cells is generally accompanied by caspase-8-dependent, delayed
ceramide production (after several hours) owing to the hydrolysis of plasma
membrane sphingomyelin as a consequence of phospholipid scrambling. Increases
in ceramide levels followed the kinetics of nuclear fragmentation and occurred
after cytochrome c release or caspase-3 activation. In addition, Raji B-cells,
which do not produce ceramides upon Fas activation, display apoptotic features
in mitochondria and the nucleus (Tepper et
al., 1997
; Tepper et al.,
2000
). These observations argue strongly against a role for
ceramides in triggering apoptosis. Also, data obtained using aSMase knock-out
mice and overexpression of a nSMase have failed to implicate these SMases in
either Fas-induced ceramide production or apoptosis of B- and T-cells
(Brenner et al., 1997
;
Cock et al., 1998
;
Tepper et al., 2001
). Thus,
although evidence abounds suggesting that ceramide can promote apoptosis, the
extent to which ceramide formation is essential remains controversial and
appears to depend largely on the cellular system used
(Cifone et al., 1995
;
Hannun et al., 1996
;
Hofmann and Dixit, 1998
).
Here we investigated some of these questions, with a focus on A20 B- and
Jurkat T-lymphoma cells, where cell death is readily induced with recombinant,
soluble FasL. We observed that FasL stimulated two distinct types of
caspase-8-dependent cell death: apoptosis that occurred via activation of
caspase-3 and necrosis which, like phosphatidylserine (PS)-externalization,
was caspase-3 independent. On the other hand, FasL also significantly
increased ceramide levels after 3 hours in A20, as previously described for
Jurkat T-cells. Treatment with cell-permeable ceramides or bacterial SMase led
to necrosis in both cell types. In Raji B-cells, lacking ceramide production
owing to absence of lipid scrambling
(Tepper et al., 2000), Fas
activation triggered only apoptosis at all antibody concentrations tested,
whereas cell-permeable ceramides and bacterial SMase promoted necrosis. In the
presence of FasL, addition of cell-permeable ceramides only promoted necrosis
in A20 and Jurkat cells when added within the first 4 hours after FasL. Thus,
Fas-induced lipid scrambling and delayed elevation of intracellular ceramide
levels are suggested to promote necrosis in cells where FasL failed to trigger
caspase-3-dependent apoptosis.
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Materials and Methods |
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Cell culture
A20, A20R and M12 cells (kindly provided by Jürg Tschopp, Institute of
Biochemistry, University of Lausanne, Switzerland) were cultured in DMEM
supplemented with 10% fetal calf serum, antibiotics (10,000 U/ml Penicillin,
10 µg/ml streptomycin) and 50 µM ethanol-2-thiol at 37°C and 5%
CO2. Ramos, Daudi and Raji human B-lymphoma lines and the
EBV-transformed human B-lymphoblast cells GES and LM, all provided by
Maria-Rosa Bono (Faculty of Basic Sciences, University of Chile), were
cultured in RPMI supplemented with 10% fetal calf serum and antibiotics at
37°C, 5% CO2.
In vitro caspase-3/caspase-1 assay
A20 and Jurkat cells (0.5x106 cells) were treated with
FasL for 0-8 hours at 37°C. To test the effect of C6-ceramide or caspase
inhibitors, cells were preincubated for 30 minutes. Then caspase activity was
measured using a previously described protocol
(Hetz et al., 2002) modified
from Boldin et al. (Boldin et al.,
1996
).
In situ caspase-3/caspase-8 assay
Caspase activity was detected using the caspase-3 substrate FAM-DEVD-fmk
(Promega, Madison, WI) or the caspase-8 substrate FAM-LETD-fmk (Intergen, New
York, NY), following instructions from the manufacturer, by flow cytometry
(FACS; Becton Dickinson, Mountain View, CA) and the Cell Quest program.
Viability assays
Cell viability was analyzed by FACS as described before
(Hetz et al., 2002). A20 cells
were incubated with either C6-ceramide or dihydro-C6-ceramide (DH-C6-ceramide)
at the concentrations indicated for up to 16 hours at 37°C. For inhibition
experiments, cells were preincubated for 30 minutes without or with the
caspase inhibitors Ac-DEVD-cho (100 µM), Ac-YVAD-cho (100 µM) or
zVAD-fmk (10 µM). Then FasL was added, and cells were incubated for another
16 hours at 37°C. Cells were harvested and stained with 10 µg/ml of
propidium iodide (PI) for determination of cell viability. For DNA content
analysis cells were permeabilized with methanol and stained with PI. Samples
containing roughly 1x104 cells were analyzed by FACS using
the Cell Quest program. Alternatively, cells were sorted by FACS using PI
fluorescence emission as the parameter for selection and nuclear morphology
was analyzed by confocal microscopy as described below.
Cell viability was also quantified by alternative methods using the reagents 3-(4,5-dimethylthazol-2-yl)-5-3-carboxymethoxy-phenyl)-2-4(sulfophenyl)-2H-tetrazolium (MTS) and phenazine methosulfate (PMS) according to the recommendations of the supplier (Promega, CellTiter96® Aqueous).
Diacylglycerol kinase assay
Ceramide concentrations were determined in 5x106 cells as
previously described (Preiss et al.,
1987; Walsh and Bell,
1986
) using brain ceramide (Avanti Polar Lipids, Alabaster, AL) as
a standard. Determinations were done in triplicate in each experiment. On
average (n=6), basal ceramide and diacylglycerol levels in A20 or
A20R cells were 2.9±1.0 pmol/nmol lipid phosphate (or 132±55
pmol per 5x106 cells) and 14±3.2 pmol/nmol lipid
phosphate (or 630±184 pmol per 5x106 cells),
respectively. These values are referred to as 100%.
DNA fragmentation assay
A20 cells (1x106/ml) were incubated in complete medium
with FasL, C6-ceramide, dihydro-C6-ceramide or combinations thereof for 4
hours at 37°C. For the inhibition experiments, cells were preincubated for
30 minutes with caspase inhibitors. Subsequently, cells were harvested by
brief centrifugation and lysed by addition of 100 µl
phenol/chloroform/isoamylalcohol (25:24:1) and centrifuged. Then, 17.5 µl
of the aqueous phase were mixed with 2 µl 10x buffer H (50 mM
Tris-HCl pH 7.5, 10 mM MgCl2, 100 mM NaCl, 1 mM dithioerythrol) and
0.5 µl RNase A solution (500 mg/ml), incubated for 30 minutes at 37°C
and analyzed by electrophoresis on a 2% agarose gel containing 0.5 mg/ml
ethidium bromide. DNA bands were visualized by exposure to UV light.
Assessment of chromatin condensation and morphological changes
Cells were treated as described previously (viability assays) and stained
after 16 hours cells with PI (1 µg/ml) for 5 minutes. After washing twice
in PBS, cells were treated with glycerol-DABCO and viewed by an SLM-400 Carl
Zeiss confocal microscopy upon excitation at 543 nm using a 570 nm emission
filter (UACI, ICBM, University of Chile). As a control, cells were
permeabilized by addition of 500 µl ice-cold ethanol and incubated for 10
minutes at -20°C before staining with PI. A total of 200 cells were
analyzed. Alternatively, cells were washed in PBS and fixed with 3%
glutaraldehyde, 100 mM Na-cacodylate for 1.5 hours at 4°C. After
post-fixation in 1% AsO4 and dehydration, cells were embedded in
EPSON 812 resin. Sections were stained with uranyl acetate and lead citrate
and were observed in a Zeiss TEM 109 Electron Microscope (Electron Microscopy
Center, ICBM, Department of Morphology, University of Chile).
Quantification of phosphatidylserine exposure
Presence of phosphatidylserine in the outer leaflet of the plasma membrane
was detected following instructions of the manufacturer by flow cytometry
using FITC-coupled annexin V (annexin V-FITC) and 5x103 cells
per experiment.
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Results |
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Addition of 100 µM of the caspase-3 inhibitor Ac-DEVD-cho following FasL treatment increased cell viability by 30%, whereas incubation with 100 µM of the caspase-1 inhibitor Ac-YVAD-cho did not protect A20 cells. Pre-incubation of cells with the broad-range caspase inhibitor zVAD-fmk (10 µM) completely protected cells against FasL-induced cell death (Fig. 1A, see below Fig. 5). These observations are consistent with the interpretation that activation of caspase-8, an initiator caspase upstream of caspase-3, represents an early event triggered by Fas, leading to cell death. Interestingly, inhibition of caspase-3 and related caspases only partially restored cell viability upon treatment with FasL.
|
|
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A hallmark of apoptosis is the generation of DNA fragments of defined length, which lead to a ladder-like pattern after separation by size. Also in A20 B-lymphoma cells, incubation with FasL led to DNA laddering (Fig. 1C), and this effect was concentration dependent (data not shown). Pretreatment of cells with the caspase-3 inhibitor Ac-DEVD-cho or the broad-spectrum caspase inhibitor zVAD-fmk reduced DNA degradation or blocked it, respectively. By contrast, Ac-YVAD-cho did not protect A20 cells from Fas-induced DNA degradation (Fig. 1C). PS exposure on the cell surface was determined by flow fluorocytometric analysis of annexin-V-FITC binding. Appearance of PS on the cell surface, observed for about 80% of the cells after treatment with 100 ng/ml FasL for 5 hours, was inhibited by zVAD-fmk but not by Ac-DEVD-cho (Fig. 1D) or Ac-YVAD-cho (data not shown). Thus, by several criteria, caspase-3 was implicated in the execution of apoptosis in FasL-stimulated A20 cells; however, FasL-induced cell death could only be partially blocked by the caspase-3 inhibitor Ac-DEVD-cho, suggesting the existence of an alternative caspase-3-independent pathway leading to A20 cell death.
Ac-DEVD-cho treatment of A20 B-lymphoma cells revealed an alternative
death signaling pathway originating from Fas
Cell viability after FasL treatment was investigated by an alternative
method using propidium iodide (PI), which labels DNA and permits the detection
of cells that have lost their membrane integrity
(Hetz et al., 2002). In the
absence of FasL, no or little A20 cell death was detectable and, as a
consequence, cells were only poorly PI positive
(Fig. 2A, NT). Upon addition of
100 ng/ml FasL (Fig. 2A, FasL),
an increase in staining was visible for a majority (90%) of the cells,
corresponding to the population found previously to be nonviable
(Fig. 1A). Surprisingly, the
dead cell population was heterogeneous, being composed of hypodiploid (M1) and
normodiploid (M2) cells. In the presence of Ac-DEVD-cho, only the hypodiploid
population was reduced, whereas with zVAD-fmk, cell viability in general was
maintained and neither dead cell population was detectable
(Fig. 2A, DEVD+FasL and
zVAD+FasL, respectively). Quantification of several such experiments
(Fig. 2B) showed that upon FasL
treatment roughly 60% of the dead cells were hypodiploid, whereas the
remaining 30% possessed normal DNA content. Ac-DEVD-cho reduced the
hypodiploid population from 60 to 35% but had no effect on dead cells with
normal DNA content. Ac-YVAD-cho had no effect at the same concentration on
either dead cell population observed in response to FasL, whereas zVAD-fmk
eliminated both.
PI binding was also analyzed in permeabilized cells, where hypodiploid (apoptotic) cells were readily distinguishable from the rest (Fig. 2C). Addition of FasL (100 ng/ml) increased the hypodiploid population by 50%. This increase was substantially reduced by Ac-DEVD-cho and completely eliminated by zVAD-fmk. Taken together, these results suggested that FasL triggered apoptotic cell death in A20 cells via a caspase-3-dependent mechanism. However, a significant fraction of the cells (about 30%) died in a distinct fashion, requiring initiator caspases but not caspase-3 activation and occurring in the absence of characteristics of apoptosis.
Morphological analysis of cells treated with FasL
To further characterize this alternative Fas-mediated pathway, cell death
observed in A20 cells in response to treatment with 100 ng/ml FasL for 16
hours was also characterized by electron microscopy
(Fig. 3A-C). Predominantly two
cell populations were detectable in response to FasL
(Fig. 3B,C). In one population,
signs of apoptosis, such as nuclear fragmentation, chromatin condensation and
membrane blebbing were observed (Fig.
3B), whereas the other was characterized by the presence of
disrupted nuclei and many vacuolar structures
(Fig. 3C). In addition, the
nuclear and cellular morphology after 16 hours of treatment with FasL were
analyzed. Nuclear morphology was revealed by PI fluorescence using confocal
microscopy (Fig. 3D). Upon
treatment with FasL (100 ng/ml), 58±10% shrunken dead cells with
condensed chromatin and a 1.5-fold reduced cell volume (A: apoptotic cells)
were visible. FACS analysis following PI staining confirmed that the
hypodiploid cell population was indeed smaller than non-treated cells (data
not shown). Additionally, FasL induced approximately 37±8% swollen dead
cells with normal nuclear staining and a roughly three-fold increased cell
volume. This second type of FasL-induced cell death is referred to from here
on as necrosis (N, necrotic cells).
|
|
A question arising at this point was whether a homogeneous A20 cell population was responding in two different ways to FasL or whether the behavior of two distinct A20 cell subpopulations was being analyzed: one that died via apoptosis and a second that died by necrosis. To address this issue, several clones (n=6) were isolated from the parent population by serial dilution and subsequently tested for their response to FasL (100 ng/ml). For all clones exactly the same pattern of cell death was observed as documented for the parent cell population (Fig. 2A,B), namely roughly 60% apoptosis versus 30% necrosis (data not shown). Thus, the A20 cells characterized here represent a homogeneous cell population that respond to FasL in two biochemically and morphologically distinguishable ways.
In situ caspase-3 and caspase-8 activity in FasL-treated A20
cells
Apoptosis is generally accompanied by a reduction in cell volume. Thus,
this parameter was employed to discriminate between apoptotic and necrotic
cells (Fig. 4A) and determine
caspase activity in situ at time points when cell viability was still
preserved (Fig. 4B). In
addition, the subpopulations were isolated by FACS sorting and characterized
in terms of their nuclear morphology (Fig.
4C) and evidence for DNA fragmentation
(Fig. 4D). Cell-permeable
fluorogenic substrates, which become fluorescent upon cleavage by caspases,
were employed for caspase-3 (FAM-DEVD-fmk) and caspase-8 (FAM-LETD-fmk)
activity. Treatment of A20 cells with FasL for 4 hours led to cell shrinkage
(Fig. 4A, region R2), which was
accompanied by caspase-3 activation (Fig.
4B) in approximately 65% of cells. No caspase-3 activation was
detected in the remaining population (Fig.
4B, region R1). Interestingly, caspase-8 activity was detected in
both populations of cells (Fig. 4B, R1 and
R2) and was completely blocked by the pre-treatment with 5 µM
of zVAD-fmk (data not shown). Furthermore, in the analysis of the nuclear
morphology after FACS cell sorting of the cell population with reduced cell
volume (R2), in which caspase-3 activation was observed, nuclear condensation
and fragmentation (Fig. 4C, R2)
were detected, although this was not the case for the rest of the cells that
died by necrosis (Fig. 4C, R1).
Electrophoretic analysis of DNA (Fig.
4D), isolated from the two subpopulations (R1 and R2), revealed
DNA fragmentation, akin to that observed for the entire population (F), only
in the R2 but not in the R1 subpopulation.
Comparative analysis of Fas-induced cell death in A20 B-, Jurkat T-
and Raji B-cells
To increase the relevance of these observations described for A20 cells,
additional models were sought. Jurkat T-cells and Raji B-cells were selected
because of their susceptibility to Fas-dependent apoptosis. The ability of
FasL (A20, Jurkat) or anti-Fas antibodies (Raji) to induce apoptosis and/or
necrosis was assessed at different concentrations
(Fig. 5A). Both apoptosis and
necrosis were found to occur side-by-side at even the lowest concentrations of
FasL tested and increased up to concentrations of roughly 100 ng/ml in A20 B-
and Jurkat T-cells. Thereafter, as FasL concentrations increased further, a
decline in necrosis was detectable. For A20 B- and Jurkat T-cells, results
similar to those illustrated for FasL were also obtained using the anti-Fas
antibody (data not shown), while the same treatment of Raji B-cells triggered
a concentration-dependent increase in apoptosis only
(Fig. 5A). Interestingly, very
low concentrations of zVAD up to 1 µM selectively reduced Fas-induced
apoptosis without affecting necrosis in both A20 and Jurkat cells
(Fig. 5B). Taken together,
these results suggest that Fas-dependent activation of initiator caspases
above a threshold value may be necessary to trigger caspase-3 dependent
apoptosis. For Jurkat cells, as previously shown with A20 cells
(Fig. 4), activation of
caspase-3 only occurred in cells of reduced volume (apoptosis), whereas
caspase-8 activation was essentially detectable in all cells (data not shown).
Thus, caspase-8 activation was a generic event occurring downstream of Fas in
A20 and Jurkat cells, whereas caspase-3 activation was only observed in the
subset of cells committed to apoptosis.
Ceramide generation after FasL stimulation
Previous reports have shown that, upon Fas ligation, lipid second
messengers like ceramides are produced in lymphoid cells owing to the
hydrolysis of plasma membrane sphingomyelin as a consequence of lipid
scrambling (Brenner et al.,
1997; Cock et al.,
1998
; Tepper et al.,
1997
; Tepper et al.,
2000
; Tepper et al.,
2001
). This phenomenon is reflected in PS exposure on the cell
surface. Interestingly, we found that PS externalization occurred in both
apoptotic and necrotic cells as evidenced by elimination of Annexin-V staining
on most FasL-treated A20 cells in the presence of zVAD
(Fig. 1D). Similar results were
also obtained with A20 and Jurkat T cells in experiments analyzing the
apoptotic and necrotic cell populations by FACS and comparing Annexin-V
staining versus cell volume following FasL treatment. PS exposure was detected
in both populations of cells. As predicted from the literature, no Annexin-V
staining was detectable for Raji cells following Fas ligation
(Fig. 5C).
Intriguingly, results in Fig.
5 indicated that Fas-induced necrosis may be linked to lipid
scrambling and PS exposure because such events do not occur in Raji B-cells.
Thus, levels of the lipid second messenger ceramide were measured in A20 cells
using the diacylglycerol kinase assay
(Preiss et al., 1987;
Walsh and Bell, 1986
). Upon
treatment of A20 cells with FasL, ceramide levels increased noticeably and
were at least two-fold above basal levels after 8 hours, whereas no such
changes were detected in diacylglycerol levels
(Fig. 6A). No significant
changes in ceramide levels were detectable within the first hour of
stimulation when A20 was compared with FasL-insensitive A20R cells (data not
shown). Fluctuations in ceramide levels observed at the 3 hour time point were
often within the range of fluctuations observed in A20R cells. On average
(n=6 or more determinations), FasL treatment increased ceramide
levels to 132±28 and 212±47 percent of baseline values, after 3
hours and 8 hours, respectively (Fig.
6B). Moreover, the kinetics of ceramide release described here
were similar to those previously reported in response to Fas activation for
Jurkat T-cells and U937 promyelocytic leukemia cells
(Sillence and Allan, 1997
;
Tepper et al., 1997
;
Tepper et al., 2000
). Thus,
Fas-induced necrosis was observed in A20 and Jurkat cells where lipid
scrambling, PS exposure and delayed ceramide increases occurred, but not in
Raji cells lacking such events.
|
Cell-permeable C2- and C6-ceramides, but not the corresponding
dihydro-ceramides, induced non-apoptotic death of A20 B-lymphoma cells
Intracellular release of ceramide upon FasL stimulation was detected either
concomitantly with or after caspase-3 activation and onset of DNA
fragmentation, suggesting that ceramide was unlikely to be mediating
apoptosis. To evaluate whether and how ceramide might contribute to cell
death, A20 cells were treated with the cell-permeable ceramide analogues
N-acetyl-(C2-ceramide) and N-hexanoyl-sphingosine (C6-ceramide). C6-ceramide
treatment for 16 hours reduced A20 viability in a concentration-dependent
manner, whereas dihydro-C6-ceramide (DH-C6-ceramide) had no such effect
(Fig. 7A). DH-C6-ceramide is
similar to C6-ceramide but lacks a critical 4-trans double bond in the
sphingosine backbone that is linked to biological activity of ceramides.
Similar reductions in cell viability were also observed upon cell treatment
with C2-ceramide, whereas DH-C2-ceramide had no effect (data not shown). Also,
incubation of A20 B-lymphoma cells with 100 µM dioctanoylglycerol, a
short-chain cell-permeable diacylglycerol analog, did not affect A20 cell
viability (data not shown). Analysis of cell viability after C2 and
C6-ceramide treatment by the PI exclusion method revealed that cells died with
no or little alteration in DNA content as assessed both by DNA laddering
(Fig. 7C) and flow cytometric
analysis (Fig. 7D,E),
suggesting that cell death occurred predominantly by necrosis. In agreement
with this interpretation, cell death induced by cell-permeable ceramides was
not associated with caspase-3 activation
(Fig. 7B) and was not inhibited
by Ac-DEVD-cho, Ac-YVAD-cho or zVAD-fmk (data not shown). Similar observations
were also made in the presence of brain ceramides (predominantly
C18-ceramides), although higher concentrations around 200 µM were required
to reduce cell viability by 50% (data not shown). Likewise, necrosis was
observed for different human B-lymphoma cell lines (Raji, Ramos, Daudi and
M12), EBV-transformed human B-lymphoblast cells (GES and LM) as well as Jurkat
cells upon treatment with cell-permeable ceramides (data not shown and
Fig. 7E).
|
Modification of endogenous ceramide levels by treatment with
bacterial SMase
To further analyze the role of ceramide in Fas-induced cell death,
endogenous ceramide levels were increased by treating cells with the
recombinant Staphylococcus aureus SMase. Such treatment of A20 cells
increased cellular ceramide levels (data not shown) and reduced cell viability
in a dose-dependent fashion (Fig.
7F). Interestingly, analysis of PI uptake and DNA content
(Fig. 7G) suggested that cells
died predominantly by necrosis. As expected, A20 cell death triggered by
bacterial SMase occurred in the absence of caspase-3 activation (data not
shown). In conclusion, Fas-induced apoptosis and ceramide-induced cell death
were distinct events in A20, Jurkat and Raji cells; however, necrosis observed
in response to FasL resembled cell death triggered by the cell-permeable C2-
and C6-ceramide analogs, brain-ceramides or treatment with bacterial
SMase.
FasL-induced caspase-3 activation, cell shrinkage and onset of DNA fragmentation were apparent within the first 2-3 hours of FasL (100 ng/ml) addition. Significantly, elevated ceramide levels, however, were observed at later time points. This raised the possibility that intracellular ceramide production may serve as a mechanism permitting A20 cells to die by necrosis in cells where caspase-3 activation either did not occur or was insufficient to trigger apoptosis. In agreement with this hypothesis, concomitant addition of C2-ceramide with FasL resulted almost exclusively in necrotic cell death after 16 hours (Fig. 8A). As ceramide addition was delayed, the ratio of apoptotic to necrotic cell death became increasingly reminiscent of the pattern observed with FasL alone (Fig. 8A), suggesting that the time point at which ceramide levels were elevated in this experimental system may serve to define the ultimate nature of cell death observed. Quite remarkably, simultaneous addition of C6-ceramide and FasL did not prevent caspase-3 activation (Fig. 7C), indicating that ceramide promoted events lying on a pathway parallel to caspase-3 activation. Similar results were also obtained with Jurkat T-cells (data not shown). Taken together these observations suggest that Fas-dependent elevation of endogenous ceramide in A20 and Jurkat cells may be delayed in order to induce necrosis only in those cells not committed to apoptosis via caspase-3 activation. Interestingly, the addition of cell-permeable ceramides to Raji cells 4-6 hours after triggering Fas also no longer altered the degree of apoptosis detected (Fig. 8B). The elevated levels of necrosis observed in this case reflect the fact that only about 50% of the Raji cells died upon Fas ligation (see Fig. 5A), suggesting that Raji cells not committed to death via Fas remained sensitive to ceramide. Thus, in all three lines characterized, cell-permeable ceramides only altered the apoptotic fate of Fas-stimulated cells when added within the first 4-6 hours following Fas activation.
Morphological analysis of cells treated with cell permeable
ceramide
The nuclear and cellular morphology after C2-ceramide treatment were
determined in A20 cells (Fig.
9). Nuclear morphology of dead cells was revealed by PI
fluorescence using confocal microscopy after 16 hours of treatment
(Fig. 9A). In cells treated
with C2-ceramide neither chromatin condensation nor cell shrinkage were
observed. Instead, ceramide-induced dead cells were two- to three-fold larger
than untreated cells. Cell death observed in A20 cells in response to
treatment with 100 µM C2-ceramide for 16 hours was also characterized by
electron microscopy (Fig.
9B,C). In perfect agreement with our other results, cells treated
with C2-ceramide (Fig. 9C)
resembled very strongly the necrotic cell population
(Fig. 3C) observed in response
to FasL treatment. Thus, by the criteria investigated, ceramide-induced death
strongly resembled FasL-induced necrosis.
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Discussion |
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The initiation of signaling pathways via Fas that lead to both apoptotic
and necrotic cell death has been described in some experimental systems. In
murine L929 fibrosarcoma cells, necrosis was observed downstream of Fas when
caspase activation was blocked by inhibitors
(Vercammen et al., 1998a).
Also, caspase inhibition rendered murine L929 fibrosarcoma cells 1000-fold
more sensitive to necrosis induced by TNF-
(Vercammen et al., 1998b
).
FasL-induced necrosis in the absence of caspase activation owing to activation
of receptor-interacting protein (RIP) has also been described previously
(Holler et al., 2000
). These
results suggest that necrosis is either favored when pathways normally leading
to apoptosis are blocked or when an alternative, caspase-independent pathway
is triggered. Our results extend such observations by showing that apoptosis
and necrosis require caspase activation and may be triggered in the absence of
caspase inhibitors. In fact, low concentrations of zVAD (less than 1 µM)
were employed to selectively reduce Fas-induced apoptosis without modulating
necrosis, whereas at higher concentrations both modes of cell death are
reduced (Fig. 5B). Thus,
elevated levels of initiator caspase-8 activity appear to favor FasL-induced
apoptosis and, as a pre-requisite, caspase-3 activation.
Analysis of ceramide levels after Fas stimulation revealed minor
fluctuations at early time points (up to 3 hours). However, the intracellular
ceramide concentrations doubled on average roughly 8 hours after stimulation
with FasL (Fig. 6A,B). Our
observations in A20 cells are consistent with previous results showing that
Fas-induced apoptosis of lymphoid cells is accompanied by a late phase of
caspase-dependent ceramide production
(Sillence and Allan, 1997;
Tepper et al., 1997
),
coinciding temporally with events occurring after caspase-3 activation, such
as nuclear fragmentation.
Recently, caspase-8-dependent but caspase-3-independent lipid scrambling
and late production of ceramide have been reported in Jurkat T cells. Neither
of these changes were detected in Raji B-cells in response to Fas activation
(Tepper et al., 2000). Our
results identified A20 B-cells as being similar to Jurkat T cells in three
ways: first, PS externalization was detectable in essentially all A20 cells
committed to death (apoptotic and necrotic cells) within the first 5 hours
after addition of FasL (Fig.
5C) and was blocked with low concentrations of zVAD-fmk but not
with caspase-3 inhibitors (Fig.
1D). Second, delayed ceramide production with similar release
kinetics was detectable (Fig.
6) (Tepper et al.,
2000
). Third, in both lines FasL triggered apoptosis and necrosis,
albeit in a distinct fashion with respect to their concentration dependence
(Fig. 5). Raji B-cells, in
contrast, only died by apoptosis (Fig.
5). Thus, our observations, in conjunction with previously
published results (Tepper et al.,
2000
), provide a link between Fas-induced lipid scrambling, late
ceramide production and cell death by necrosis.
Delayed ceramide production was additionally linked to FasL-induced
necrosis since cell-permeable C6- or C2-ceramide induced cell death
(LD5040 µM after 16 hours) without caspase-3 activation,
DNA fragmentation, cell shrinkage and chromatin condensation (Figs
7 and
9). Instead, cells increased in
size (Fig. 9A) and were filled
with vacuolar structures (Fig.
9C), resembling FasL-induced cell death by necrosis (see
Fig. 3C-E). Likewise, increases
in endogenous ceramide levels upon treatment with bacterial SMase also
promoted cell death by necrosis (Fig.
7F,G). Experiments in which ceramides were added to A20 cells at
different time points after stimulation with FasL identified the
ceramide-effect as being dominant in the sense that the earlier ceramide was
present, the higher the percentage of cell death via necrosis. However, once
the apoptotic program had been initiated, exogenous addition of ceramides no
longer had any effect, and execution of apoptosis proceeded normally
(Fig. 8A). Interestingly,
similar effects were observed for Jurkat (not shown) and Raji cells
(Fig. 8B), indicating that the
kinetics of events leading to apoptosis were well conserved between cell lines
and that delayed elevation of ceramide by exogenous supplementation had
remarkably similar consequences.
Sphingomyelin, initially located in the outer leaflet of the plasma
membrane, accumulates in the inner leaflet as a consequence of lipid
scrambling, and this is reflected in PS externalization. Hydrolysis in the
inner leaflet by a neutral SMase is then held responsible for late ceramide
production (Tepper et al.,
2000). For Raji B-cells lacking lipid-scrambling activity,
ceramide levels do not increase in response to Fas activation and, as a
consequence, substrate availability is proposed to represent a rate limiting
step in activation of the neutral SMase responsible
(Tepper et al., 2000
). Raji
cells, unlike A20 B- and Jurkat T-cells, did not undergo Fas-induced necrosis,
supporting the notion that Fas-induced delayed ceramide increases promote
necrosis. Consistent with this interpretation, addition of cell-permeable
ceramides and treatment of cells with bacterial SMase triggered necrosis.
However, experiments to implicate further one or the other intracellular
pathway known to regulate ceramide levels were unsuccessful. In our hands,
none of the compounds that reportedly modulate either neutral SMase [reduced
glutathione, L-buthionine-[S,R]-sulfoximine:
(Liu et al., 1998
)) or acidic
SMase (Imipramine: (Strelow et al.,
2000
)] activity or ceramide biosynthesis [Fumonisin B1:
(Blazquez et al., 2000
)]
altered significantly Fas-induced necrosis (data not shown). Thus, our
experiments have so far not provided a link at the molecular level to connect
lipid scrambling, delayed ceramide increases and necrosis. Further experiments
are needed to address such issues.
Several reports have suggested that ceramide production participates in
apoptotic cell death, mainly by correlating the simultaneous appearance of
apoptotic markers with ceramide production
(Garcia-Ruiz et al., 1997;
Gudz et al., 1997
). However,
experiments in which genetic manipulation was employed to analyze the
contribution of different SMases in Fas-induced apoptosis failed to implicate
any of these enzymes (Brenner et al.,
1997
; Cock et al.,
1998
; Tepper et al.,
2001
). In our experimental system, a small subpopulation of
hypodiploid cells was observed by the FACS analysis after ceramide treatment
(Fig. 7D). These most probably
represent apoptotic cells. Others have reported induction of apoptosis in a
small fraction of A20 cells (roughly 15%) by cell-permeable ceramide after 16
hours of incubation (Bras et al.,
2000
). The amount of necrosis induced by ceramide treatment
appears to depend on the cell line investigated. For instance, in the human
colon carcinoma cell line HT29, ceramide at 20 µM induces cell death in
roughly 30% of the population and only about half of those cells die by
apoptosis (C.A.H. and A.F.G.Q., unpublished). Since the assays generally
employed are not quantitative and are heavily geared towards detecting
apoptosis, it is conceivable that information concerning other forms of cell
death might have gone largely unperceived.
In contrast to apoptosis, cell death by necrosis is typically associated
with inflammation. This difference is related to activation or maturation of
phagocytic cells, like macrophages and dendritic cells
(Fadok et al., 2000;
McDonald et al., 1999
;
Sauter et al., 2000
).
Recently, Bhardwaj and coworkers showed that immature dendritic cells
phagocytose a variety of apoptotic and necrotic cells
(Sauter et al., 2000
).
However, only exposure to necrotic cells provided the signals required for
dendritic cell maturation, resulting in upregulation of maturation-specific
markers, co-stimulatory molecules and the capacity to induce antigen-specific
CD4+ and CD8+ T-cells. Thus, dendritic cells are able to
distinguish between the two types of dead cells and respond in a distinct
manner. Although the interaction between apoptotic and phagocytic cells
induces an anti-inflammatory response
(Fadok et al., 2000
), necrosis
appears to be critical for initiation of an immune response
(Holler et al., 2000
;
Sauter et al., 2000
).
Interestingly, the results presented here reinforce the notion that lipid
scrambling and PS externalization do not provide the molecular basis to
distinguish between cells dying by apoptosis or necrosis and, as a
consequence, elicit different responses of the immune system.
Simultaneous or delayed activation of pathways leading to necrotic and
apoptotic death in the same cells is likely to occur fairly frequently
(Ankarcrona et al., 1995;
Barros et al., 2001
;
Dypbukt et al., 1994
;
Hetz et al., 2002
;
Jonas et al., 1994
). It has
been proposed that the modification of intracellular ATP concentrations may
serve as one possible mechanism permitting the switch from apoptotic to
necrotic cell death (Leist et al.,
1997
). Indeed, ceramides have been shown to directly modulate
mitochondria function, for instance, by inhibiting the mitochondrial
respiratory complex III (Garcia-Ruiz et
al., 1997
; Gudz et al.,
1997
; Quillet-Mary et al.,
1997
). Thus, ceramide-induced necrosis may result from a decrease
in ATP levels due to mitochondrial dysfunction. Consistent with this notion, a
decrease in mitochondrial membrane potential was detected in A20 cells after
at least one hour of incubation with cell permeable ceramides (data not
shown). However, further experiments are required to determine how ceramide
promotes necrosis in the cells characterized here.
In summary, the results presented show that two different pathways emerge from the Fas-receptor, one leading to caspase-3-dependent apoptosis and the other favoring necrosis in a manner dependent upon activation of caspase-8, but not execution caspases, like caspase-3. In addition, Fas-dependent, delayed production of ceramide was observed. The evidence available suggests that the time point at which ceramide levels were substantially elevated dictated the extent to which necrosis was observed. Thus, Fas-induced ceramide release is proposed to permit cells to undergo necrosis when caspase-8 activation occurred but was insufficient to trigger caspase-3-dependent apoptosis. Ceramide production in A20 B-cells and Jurkat T-cells downstream of caspase-8 may be temporally delayed to trigger necrosis only in those cells not already committed to apoptosis.
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