Gastroenterology Division, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02215
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ABSTRACT |
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Apoptosis is a
physiological cell death that culminates in mitochondrial permeability
transition and the activation of caspases, a family of cysteine
proteases. Necrosis, in contrast, is a pathological cell death
characterized by swelling of the cytoplasm and mitochondria and rapid
plasma membrane disruption. Necrotic cell death has long been opposed
to apoptosis, but it now appears that both pathways involve
mitochondrial permeability transition, raising the question of what
mediates necrotic cell death. In this study, we investigated mechanisms
that promote necrosis induced by various stimuli
(Clostridium difficile toxins,
Staphylococcus aureus alpha toxin,
ouabain, nigericin) in THP-1 cells, a human monocytic cell line, and in monocytes. All stimuli induced typical features of necrosis and triggered protease-mediated release of interleukin-1 (IL-1
) and
CD14 in both cell types. K+
depletion was actively implicated in necrosis because substituting K+ for
Na+ in the extracellular medium
prevented morphological features of necrosis and IL-1
release.
N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone, a broad-spectrum caspase inhibitor, prevented morphological features of necrosis, plasma membrane destruction, loss of
mitochondrial membrane potential, IL-1
release, and CD14 shedding
induced by all stimuli. Thus, in monocytic cells, necrosis is a cell
death pathway mediated by passive
K+ efflux and activation of
caspase-like proteases.
apoptosis; toxin; N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone; interleukin-1; CD14
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INTRODUCTION |
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APOPTOSIS IS A CELL DEATH pathway essential for the development and maintenance of diverse cell populations in multicellular organisms (8). Many pathways of apoptosis stimulate mitochondrial permeability transition and the subsequent release of pro-apoptotic factors (cytochrome c and apoptosis-inducing factor) into the cytoplasm. These activate caspase-3, a member of the caspase family of cysteine proteases, which triggers with other caspases a specific pattern of protein and DNA degradation (14, 19, 20).
Necrosis, in contrast to apoptosis, results from cell injury and is associated with inflammation. This form of cell death is characterized by cell swelling, dilatation of mitochondria, and a gross increase in plasma membrane permeability allowing passive diffusion of ions (9, 16). Necrosis has long been considered a degenerative process completely different from apoptosis. However, recent studies have shown similarities and connections between apoptosis and at least some models of necrosis. For instance, anti-apoptotic proteins of the Bcl-2 family, which control mitochondrial osmotic homeostasis, prevent some models of necrosis (23, 27). Also, intracellular ATP depletion can shift an apoptotic response to necrotic cell death (7). These data suggest that necrosis is a regulated pathway, but little is known about its regulation.
The aim of this study was to identify mediators that promote necrosis
in human monocytes and THP-1 monocytic cells. We found that various
toxins induced a patterned necrotic cell death associated with release
of soluble CD14 and interleukin-1 (IL-1
). The data indicate that
this cell death pathway is mediated by
K+ depletion and activation of
caspase-like proteases.
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MATERIALS AND METHODS |
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Cells.
Human peripheral blood monocytes were isolated by Ficoll-Paque gradient
centrifugation and Percoll discontinuous density gradient (Pharmacia,
Piscataway, NJ) (26). Preparations contained 50-80% monocytes as
determined by CD14 expression. THP-1 cells, a human monocytic cell line
[American Type Culture Collection (ATCC), Manassas, VA],
were cultured in RPMI 1640 supplemented with 5 mM HEPES, 5% FCS,
and 50 µM -mercaptoethanol (GIBCO, Grand Island, NY).
Toxin purification. Clostridium difficile toxin A (TcdA) and toxin B (TcdB) were purified from VPI 10463 strain (ATCC) culture supernatant as previously reported (24, 29).
Cell stimulation.
Monocytes were kept in suspension by gentle agitation in glass tubes.
THP-1 cells (500,000 cells/ml) were preincubated in 100 ng/ml
lipopolysaccharide (LPS) from Escherichia coli 055:B5 (Sigma)
for 1 h to trigger pro-IL-1 synthesis. Then cells were suspended in fresh medium and stimulated with TcdA, TcdB,
Staphylococcus aureus alpha toxin
(SAT) (Sigma, St. Louis, MO), ouabain, or nigericin (Sigma) for 2 h.
Monocytes and THP-1 cells were stimulated in RPMI 1640, 5 mM HEPES, 50 U/ml penicillin G, and 50 µg/ml streptomycin. CD14 expression was
induced in THP-1 cells with 1, 25-dihydroxyvitamin D3 (100 nM for 48 h; BioMol,
Plymouth Meeting, PA) (21). The following protease inhibitors were
added 15 min before stimulation: 100 µM
N
-p-tosyl-L-phenylalanine
chloromethyl ketone (Sigma), 50 µM
N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl
ketone (z-VAD-fmk) (Calbiochem, San Diego, CA), 50 µM
N-acetyl-Tyr-Val-Ala-Asp-aldehyde or
50 µM
N-acetyl-Asp-Glu-Val-Asp-aldehyde
(DEVD-cho; BioMol). Stock solutions (10 mM) of caspase inhibitors were
prepared in DMSO. K+ buffer (10 mM) contained (in mM) 150 KCl, 5 NaH2PO4,
10 HEPES, 1 MgCl2, 1 CaCl2, and 5 glucose and
0.5% BSA.
Cell death assays and flow cytometry.
Necrosis and apoptosis were distinguished on the basis of four
independent assays: 1) morphology,
2) plasma membrane permeability to
propidium iodide, 3) DNA content,
and 4) cell surface expression of
phosphatidylserine. Control apoptotic cells were obtained by incubation
in 100 µM
N-tosyl-L-lysine
chloromethyl ketone and 25 µM cycloheximide as previously described
(31). Necrotic THP-1 cells and monocytes were identified by microscopy
on the basis of cell swelling and cytoplasm translucidity. Loss of
membrane integrity was determined by flow cytometry using propidium
iodide. Propidium iodide (10 µg/ml; Sigma) was added just before
analysis. Cell DNA content was quantified by flow cytometry using
propidium iodide (18). After stimulation, cells were successively fixed in 70% ethanol for 30 min, incubated with RNase (1 mg/ml) for 1 h, and
stained in 10 µg/ml propidium iodide, 0.1% sodium citrate, and 0.1%
Nonidet P-40. Phosphatidylserine expression was detected by flow
cytometry using FITC-annexin V (Trevigen, Gaithersburg, MD).
Mitochondrial membrane potential was measured using rhodamine 123 (Molecular Probes, Eugene, OR) as previously described (27). Cell
surface CD14 was quantified by flow cytometry using FITC-MY4 monoclonal
antibodies (Coulter, Miami, FL). Flow cytometry analysis was performed using a FACScan and the Cellquest software
(Becton-Dickinson, San Jose, CA).
IL-1 and soluble CD14 concentrations.
Mature IL-1
levels were measured by ELISA (Cistron, Pine Brook, NJ).
Soluble CD14 was also quantified by ELISA (Medgenix Diagnostics,
Fleurus, Belgium).
Intracellular
K+.
Intracellular K+ concentration
([K+]i)
was measured using the fluorescent dye
K+-binding benzofuran isophthalate
(PBFI) (13). THP-1 cells (106
cells/ml) were loaded with 5 µM PBFI for 90 min (Molecular Probes, Eugene, OR). After a washing, cells were resuspended (500,000 cells/ml)
in (in mM) 150 NaCl, 5 NaH2PO4,
10 HEPES, 1 MgCl2, 1 CaCl2, and 5 glucose and 0.5%
BSA. Cells were stimulated for 2 h at 37°C, and fluorescence was
measured using a Perkin-Elmer LS-50B spectrofluorometer (Norwalk, CT).
Excitation wavelength was set at 340 or 380 nm, the isosbestic point.
Emission was measured at 505 nm (5-nm slit width). The ratio of the
fluorescence intensities (340/380 nm) was used to determine
K+ concentration. The PBFI assay
was calibrated by equilibrating [K+]i
with known extracellular K+
concentration
([K+]e).
[K+]e
was increased from 0 to 150 mM in 50-mM increments by substituting K+ for
Na+, and cells were permeabilized
using 30 nM SAT. There was a close linear correlation between
fluorescence values and
[K+]e
(Pearson correlation coefficient 0.95).
Statistical analysis. Statistical analyses were performed using one-way ANOVA followed by the Student-Newman-Keuls test (SigmaStat, Jandel Scientific Software, San Rafael, CA).
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RESULTS |
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Necrosis of THP-1 cells and monocytes is associated with release of
CD14 and IL-1.
In preliminary experiments, we tested a series of agents for their
ability to induce monocyte necrosis. We found that TcdA (100 nM) and
TcdB (10 nM), SAT (10 µg/ml), nigericin (10 µM), and ouabain (200 µM) cause monocyte and THP-1 cell necrosis within 1.5-3 h.
Morphological features of necrosis were identical in both cell types
(Fig. 1,
A-F).
The cytoplasmic translucence associated with necrosis caused a drop in
forward scatter height as measured by flow cytometry (Fig. 1,
K-L).
The toxins used in these experiments have distinct mechanisms of
action. Briefly, TcdA and TcdB catalyze the monoglucosylation of Rho
proteins, which disrupts the actin cytoskeleton (12). SAT forms plasma
membrane pores permeable to monovalent cations (2, 3). Ouabain inhibits
the
Na+-K+
pump ATPase, whereas nigericin is a
K+ ionophore. All stimuli caused
cell swelling and permeability to propidium iodide in <2 h (nigericin
and ouabain not shown) (Figs. 1 and
2A).
In contrast to apoptotic THP-1 cells, necrotic cells did not show a
decrease in DNA content (Fig. 1,
A-D)
or phosphatidylserine exposure before loss of membrane integrity (data
not shown). In addition to causing cell necrosis, all stimuli induced
the release of mature IL-1
from LPS-stimulated THP-1 cells within 2 h (Fig. 2B). In normal monocytes
TcdB (1 nM) also induced a 16.6 ± 2.0-fold increase in IL-1
release at 30 min (means ± SE, n = 3, P < 0.05). All stimuli also caused a
marked reduction in cell surface expression of the LPS receptor CD14 in
vitamin D3 differentiated THP-1
cells (undifferentiated THP-1 cells do not express CD14; Fig.
2C). In monocytes TcdA, TcdB, and
SAT also reduced cell surface CD14 expression (by 91, 81, and 89%,
respectively). This was associated with a corresponding release of
soluble CD14 (Fig. 2,
D-E).
Thus, in normal monocytes as well as in THP-1 cells, necrosis induced
by different toxins is associated with the rapid release of IL-1
and
soluble CD14. Despite their distinct mechanisms of action, these agents
stimulate a common pattern of events, suggesting activation of common
mediators of necrosis.
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Blocking K+
efflux prevents monocyte necrosis and IL-1 release.
In many cell types, both SAT and ouabain cause
K+ depletion as a direct
consequence of their mechanism of action. We tested the hypothesis that
K+ depletion is involved in THP-1
cell and monocyte necrosis. In THP-1 cells all stimuli caused a
decrease in
[K+]i
in controls (140 ± 5 mM) and in cells stimulated with SAT (42 ± 5 mM), TcdB (38 ± 4 mM), nigericin (5 ± 20),
and ouabain (80 ± 4; all values are means ± SE). We next
examined the effect of blocking K+
efflux by substituting K+ for
Na+ in the extracellular medium.
In control THP-1 cells, the high K+ buffer induced discrete
cytoplasmic extensions and the subsequent incubation in normal buffer
did not trigger necrosis. The high K+ buffer prevented the
morphological changes of necrosis and IL-1
release by all stimuli
(Fig. 3,
A, B,
D, and
E; ouabain and nigericin data not
shown). The subsequent replacement of the high
K+ buffer by normal medium
triggered necrosis (Fig. 3, C and
F) and IL-1
release (Fig.
3G) within 20 min. Incubation of
monocytes in the high K+ buffer
also prevented necrosis following exposure to all necrotic stimuli
(data not shown). Furthermore, the subsequent replacement of the high
K+ buffer by normal medium
triggered necrosis in up to 40% of the control monocytes. Taken
together, these data support the hypothesis that
K+ efflux regulates monocyte
necrosis induced by various stimuli.
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z-VAD-fmk, a broad-spectrum caspase inhibitor, prevents THP-1 cell
and monocyte necrosis.
In monocytes not undergoing necrosis, inducing a net
K+ efflux activates caspase-1
(formerly IL-1 converting enzyme), leading to IL-1 release (22, 30).
In THP-1 cells undergoing necrosis, we found that
N-acetyl-Tyr-Val-Ala-Asp-aldehyde, a
specific caspase-1 inhibitor, abrogated IL-1
release by all stimuli,
but did not prevent necrosis (data not shown). We tested whether other
caspases, in addition to caspase-1, might be implicated in THP-1 cell
necrosis. Cell preincubation with z-VAD-fmk (50 µM), a broad-spectrum
caspase inhibitor, completely prevented the morphological features of necrosis in THP-1 cells and in monocytes (Fig.
4,
A-E;
in separate control experiments 0.5% DMSO, the solvent for caspase
inhibitors, did not affect necrotic cell number in control or
stimulated cells). z-VAD-fmk also prevented THP-1 cell membrane leakage
in contrast to the caspase-3 inhibitor DEVD-cho (Fig. 4,
F-I).
In addition, z-VAD-fmk inhibited IL-1
release and CD14 shedding in
THP-1 cells stimulated by all stimuli (>95% inhibition; Fig.
5). These findings indicate that key events
in monocytic cell necrosis are mediated by activation of caspase-like
proteases.
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K+ depletion
may precede or follow protease activation during monocyte necrosis.
To determine whether K+ efflux was
upstream or downstream of protease activation, we measured
[K+]i
in THP-1 cells protected from necrosis by preincubation with the
caspase inhibitor z-VAD-fmk. The pore-forming toxin SAT and the
Na+-K+
pump ATPase inhibitor ouabain both caused marked
[K+]i
depletion in z-VAD-fmk-treated cells, after 1 h of incubation. No
changes in
[K+]i
were observed in z-VAD-fmk-treated cells challenged with TcdB or
nigericin (Fig. 6). Thus agents that induce
[K+]i
depletion (e.g., SAT and ouabain) can trigger protease activation and
subsequent necrosis. However, for other stimuli (e.g., TcdB or
nigericin)
[K+]i
depletion is the result, not the cause, of protease activation.
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z-VAD-fmk prevents loss of mitochondrial membrane potential.
In the effector phase of apoptosis as well as in necrosis, mitochondria
lose osmotic homeostasis and membrane potential. We tested whether
activation of caspase-like proteases occurs upstream or downstream to
mitochondrial membrane depolarization. THP-1 cells were stained with
rhodamine 123, a fluorescent probe uptaken by mitochondria that
maintain a membrane potential. The distribution of rhodamine 123 fluorescence was biphasic in control cells, 70% of them showing a
10-fold higher signal than the remaining 30%. z-VAD-fmk preincubation
prevented the drop in rhodamine 123 fluorescence caused by all stimuli
(Fig. 7; TcdB and ouabain not shown). These findings suggest that activation of caspase-like proteases precedes mitochondrial membrane depolarization in THP-1 cells undergoing necrosis.
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DISCUSSION |
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The present study shows that necrosis of monocytes and THP-1 cells
induced by various stimuli is a cell death pathway associated with
IL-1 release and CD14 shedding. Moreover, this pathway involves caspase-like proteases and K+ depletion.
The broad-spectrum caspase inhibitor z-VAD-fmk has been shown to inhibit various pathways of apoptosis in many cell types. In THP-1 cells, z-VAD-fmk (50 µM) prevented apoptosis (31) and inhibited the cleavage of caspase-2, -3, -6, and -7 (15). z-VAD-fmk also prevented z-DEVD-AFC (benzyloxycarbonyl-Asp-Glu-Val-Asp-7-amino-4-trifluoromethyl coumarin) cleavage (a caspase-3 substrate; IC50 1.2 µM) and activation of caspase-3 (15). To our knowledge, z-VAD-fmk has not been reported to inhibit necrosis. In contrast, it was shown to promote necrotic cell death in some other cell types (9, 27), suggesting important cell type-related differences in the control of necrosis. Caspase-3, a central effector of apoptosis, does not appear to be involved in THP-1 cell necrosis because DEVD-cho did not prevent necrosis. However, we cannot exclude the possibility that DEVD-cho did not achieve sufficient intracellular concentrations to block caspase-3 activity in our experiments.
Although the target(s) of z-VAD-fmk was not identified in this study,
the data strongly suggest that monocyte necrosis involves the
activation of caspases or caspase-like proteases. In monocytes not
undergoing necrosis, IL-1 release involves caspase-1 activation, is
enhanced by K+ efflux, and is
blocked by increasing K+
concentration in the extracellular medium (22, 30). In this study, we
show that IL-1
release and necrosis are associated and that both are
inhibited by intracellular K+ and
by a broad-spectrum caspase inhibitor. These data support the
hypothesis that monocyte necrosis is mediated by activation of caspases
or other z-VAD-fmk sensitive proteases.
Early studies reported that necrotic cell death is associated with rapid loss of intracellular K+ (9). To our knowledge, this is the first report that maintaining [K+]i can inhibit cell necrosis. A role for K+ in pro-apoptotic caspase regulation was recently reported. In HeLa cells and cerebellar granules, K+ deprivation caused caspase-3 activation and apoptosis (4, 5). Moreover, K+, at a physiological intracellular concentration (150 mM), prevented recombinant caspase-1 activation in vitro (5). Although this effect was not specific to K+ (Na+, Ca2+ and Mg2+ at 150 mM were as potent as K+), these results support the hypothesis that caspase activation is prevented in healthy cells by K+, the main intracellular cation. In support of this hypothesis, apoptosis induced by various stimuli in lymphocytes was associated with progressive K+ loss. Replacement of Na+ by K+ in the extracellular medium prevented lymphocyte apoptosis and caspase-3 activation (11). The time course of K+ depletion in these lymphocytes was much slower (56 mM after 8 h) than in THP-1 cells undergoing necrosis (median value 40 mM after 2 h). The rapid loss of plasma membrane integrity that characterizes necrosis is likely to allow rapid intracellular K+ depletion and may promote necrosis versus apoptosis. Thus intracellular K+ appears to be a key inhibitor of cell death through its ability to prevent caspase activation.
The findings that monocyte necrosis involves intracellular K+ and caspase-like proteases is compatible with the emerging concept that apoptosis and necrosis may be functionally linked cell death pathways (reviewed in Ref. 14). Both modes of cell death involve loss of mitochondrial membrane potential and osmotic homeostasis, under the control of Bcl-2 proteins. It is interesting that Bcl-xL, an anti-apoptotic protein of this family, forms cation-selective membrane channels and is capable of conducting a K+ current (18). Recently, Bcl-xL was found to inhibit necrosis caused by inhibitors of oxidative phosphorylations (27). These studies provide further evidence that the regulation of intracellular cations plays a key role in both apoptosis and necrosis. Elucidating the mechanism by which intracellular K+ prevents caspase activation will represent a key step in understanding the regulation of cell death.
Based on the findings of this study and the current literature, we speculate that monocyte necrosis results from activation of K+-regulated caspases (or caspase-like proteases), which in turn cause loss of mitochondrial membrane potential and permeability transition. This pathway of cell death can be induced by marked K+ depletion or by other mechanisms independent of [K+]i. Permeability transition and loss of mitochondrial osmotic homeostasis further promote K+ loss and caspase activation by a self-amplifying mechanism involving ATP depletion (inhibition of K+ uptake) and plasma membrane disruption, two classical features of necrosis. Further studies are needed to verify this hypothesis and to identify these "pro-necrotic" caspases.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: M. Warny, Gastroenterology Div., Dana Bldg., Rm. 501, Beth Israel Deaconess Medical Center, Harvard Medical School, 330 Brookline Ave., Boston, MA 02215 (E-mail: mwarny{at}caregroup.harvard.edu).
Received 11 June 1998; accepted in final form 12 December 1998.
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