From the Department of Immunology, Scripps Research
Institute, La Jolla, California 92037 and the
Van Andel
Research Institute, Grand Rapids, Michigan 49503
Received for publication, September 10, 2002, and in revised form, November 18, 2002
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ABSTRACT |
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Macrophages from different inbred
mouse strains exhibit striking differences in their sensitivity to
anthrax lethal toxin (LeTx)-induced cytolysis. Although LeTx-induced
cytolysis of macrophages plays an important role in the outcome of
anthrax infection, the sensitivity of macrophages in vitro
does not correlate with in vivo susceptibility to infection
of Bacillus anthracis. This divergence suggests that
additional factors other than LeTx are involved in the cytolysis of
LeTx-resistant macrophages in vivo. We found that
LeTx-resistant macrophages became sensitive to LeTx-induced cytolysis
when these cells were activated by bacterial components. Tumor necrosis
factor- Anthrax infections are initiated by endospores of
Bacillus anthracis (1). These spores germinate
after they are phagocytosed by macrophages and begin to express
virulence toxins that lead to a systemic immune response, shock, and
death (1). It has been shown that the three toxins produced by anthrax
bacilli, edema factor (EF),1
lethal factor (LF), and protective antigen (PA), are principally responsible for provoking the host responses (1, 2). PA functions as a
molecular transporter by facilitating the entry of LF and EF into cells
through endocytosis and translocation of EF and LF into the cytosol (3,
4). LF, a zinc metalloprotease (4), together with PA is termed lethal
toxin (LeTx). LeTx is the major contributor of virulence in infected
animals (1, 5). EF, an adenylate cyclase (3), functions as an enhancer that enhances the cytotoxicity of LeTx in macrophages (6).
Much is known about the mechanisms of action and cellular entry of LF
and EF. PA binds to a cell-surface receptor (7), where it is
proteolytically processed on the cell surface (8, 9). The processed PA
heptamerizes and binds 3 molecules of either EF or LF, resulting in
either a PA·EF or a PA·LF complex, which is internalized through
receptor-mediated endocytosis (10). PA then forms a hole in the
acidified endocytic vesicle through which EF or LF is delivered to the
cytosol (11). Although it is known that EF exerts its effects through a
calmodulin-dependent adenylate cyclase activity, the
mechanisms of LF intoxication are less clear. LF can be delivered to
the cytosol of cells, but cytolysis as a consequence of the
intracellular LF is observed only in macrophages from certain mouse
strains (5). Conflicting results have been reported regarding whether
LeTx induces cytokine production in macrophages (12-14). The toxic
activity of LF depends on its protease activity (4), suggesting that
proteolysis of one or more cellular protein(s) unleashes a cascade of
events resulting in the death of the intoxicated macrophages. Indeed, cleavage of several mitogen-activated protein kinase (MAPK) kinases (MKKs) has been observed (15-17). However, the physiological
importance of MKK proteolysis relative to macrophage cytolysis cannot
be established because similar cleavage is observed in macrophages that
are resistant to LeTx (16, 17).
Inbred mouse strains exhibit striking differences in the sensitivity of
their cultured macrophages to the effects of LeTx (5). For example, C3H
mouse macrophages lysed by LeTx at 1 ng/ml are 100,000 times more
sensitive than A/J mouse macrophages (5). Macrophages from CBA/J and
BALB/c mice are sensitive, whereas those from C57BL/6J and
DBA/2J mice are resistant. Direct binding studies revealed that the
affinity and number of PA receptors/cell are the same in sensitive and
resistant cells (5). Proteolytic activation of PA is also the same in
both sensitive and resistant macrophages (5). Resistant macrophages are
not cross-resistant to other toxins and viruses that, like lethal
toxin, require vesicular acidification for activity. This implies that
resistance is not due to defects in vesicular acidification (5). As
mentioned earlier, the proteolysis of MKKs is comparable in sensitive
and resistant cells (16, 17). Thus, resistance is due to a defect at a
stage occurring after proteolysis. Watters et al. (17) identified a mutation on a gene named kif1C that is
responsible for the different sensitivities of macrophages to LeTx.
kif1C encodes a kinesin-like motor protein of the
UNC104 subfamily (18). It is clear that proper function of kif1C
is required for LeTx resistance, but how this protein is involved in
LeTx-elicited changes in macrophages is unknown (17). The kif1C protein
has been excluded as a target of LF-mediated proteolysis (17).
It was believed that LeTx-induced cytolysis of macrophages played an
important role in the overall outcome of anthrax infection. Injection
of LeTx into CBA/J mice, whose macrophages are sensitive to LeTx,
results in a more rapid death compared with A/J mice, whose macrophages
are resistant to LeTx-induced cytolysis (5). However, the sensitivity
of macrophages to LeTx in vitro does not correlate with
in vivo susceptibility to infection of either encapsulated
or non-encapsulated strains of B. anthracis (5, 16, 17).
Because macrophages can be activated by encounters with infected
bacteria (19, 20), we addressed whether macrophage activation has an
effect on cell viability in LeTx-treated LeTx-resistant macrophages. We
found that treatment of macrophages with bacterial components can make
LeTx-resistant macrophages become sensitive to LeTx-induced cytolysis.
Tumor necrosis factor- Materials--
Recombinant PA and LF were prepared as described
(15). Murine TNF was provided by Dr. Vladimir Kravchenko
(Scripps Research Institute). Anti-TNF antibodies were raised in
rabbits using recombinant TNF. Lipopolysaccharides, peptidoglycan, and
poly-D-glutamic acid were from Sigma. IL-1 Preparations of Peritoneal Macrophages--
Peritoneal
macrophages were obtained from mice by saline lavage as previously
described (21). Mice received intraperitoneal injections of 3%
thioglycolate 4 days before the preparation of peritoneal macrophages.
After anesthetization with intramuscular injections of a mixture
of ketamine (80 mg/kg of body weight) and xylazine (16 mg/kg of body
weight), peritoneal macrophages were harvested from the mice through
lavage of the peritoneal cavity with 5 ml of saline. The cells were
then washed with RPMI 1640 medium and resuspended in Dulbecco's
modified Eagle's medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin, 100 µg/ml streptomycin, and 2 mM
glutamine (supplemented Dulbecco's modified Eagle's medium), and
1 × 105 or 2 × 106 cells were
plated on 96-well microtiter or six-well plates, respectively. After
incubation at 37 °C for 2 h, the non-adherent cells were removed by washing three times with fresh Dulbecco's modified Eagle's
medium and cultured overnight in supplemented Dulbecco's modified
Eagle's medium.
Cell Viability Assays--
The extent of cell death was measured
using crystal violet uptake and propidium iodide exclusion assays (22).
Briefly, the cell culture medium was removed from the plates by
immersing them vertically in a beaker containing 2 liters of 0.9%
saline. After immersion, all liquid was tapped out onto paper towels.
To stain cells, 80 µl of a 5% crystal violet solution containing
25% methanol in saline was added to each well and incubated for 5 min
at room temperature. Excess stains were removed by immersing
plates twice in saline as described above. After removal of all liquid
as described above, 100 µl of 50% acetic acid was added to each well
to dissolve all the stained cellular materials. Plates were then placed
on a shaker for ~30 min, and the absorbance at 590 nm was measured. Propidium iodide staining was performed using trypsinized cells resuspended in phosphate-buffered saline with 1 µg/ml propidium iodide. The level of propidium iodide incorporation was quantified by
flow cytometry on a FACScan flow cytometer.
Hoechst 33258 and Annexin V Staining--
Nuclear condensation
and fragmentation were determined by staining the cells with Hoechst
33258 (Sigma) as described previously (22). Phosphatidylserine exposure
to the outer leaflet was analyzed by staining cells with fluorescein
isothiocyanate-conjugated annexin V (Roche Molecular Biochemicals) as
suggested by the manufacturer. Briefly, cells were resuspended in
annexin labeling solution containing 10 mM HEPES (pH 7.4),
140 mM NaCl, 5 mM CaCl2, and
fluorescein isothiocyanate-conjugated annexin V for 25 min.
After washing twice with phosphate-buffered saline, cell pellets were
resuspended in propidium iodide (1 µg/ml)-containing
phosphate-buffered saline and analyzed by flow cytometry.
DNA Ladder Assay--
Cells were collected, lysed in lysis
buffer (10 mM Tris-HCl (pH 8.0), 100 mM NaCl,
0.5% SDS, 25 mM EDTA, and 0.1 mg/ml protease K),
and incubated at 50 °C overnight. DNA was
phenol/chloroform-extracted and precipitated by adding NaCl to 0.3 M and 2 volumes of ethanol. The DNA pellets were
resuspended in Tris/EDTA buffer with 0.1 mg/ml RNase I for
1 h at room temperature and then treated with 0.1 mg/ml protease K
overnight at 37 °C. DNA was further phenol/chloroform-extracted, precipitated as described above, and resuspended in 20 µl of
Tris/EDTA buffer. Equal amounts of DNA were separated on 2% agarose
gel and stained with ethidium bromide for visualization.
Total Cell Lysate Preparation and Western Blot
Analysis--
Total cell lysate extraction and Western blot analysis
for MEK1 were performed as previously described (22). Briefly, cells were lysed in ice-cold cell lysis buffer containing 20 mM
MOPS, 15 mM EGTA, 2 mM EDTA, 1 mM
Na3VO4, 1 mM dithiothreitol, 75 mM LeTx-resistant Macrophages Are Selectively Resistant to
LeTx-induced Cell Death--
We used macrophages from C3H/HeN, 129svj,
and C57BL/6J mice to repeat published results indicating that
macrophages from different mouse strains have different
sensitivities to LeTx-induced cell killing in vitro. As
shown in Fig. 1A, macrophages
from C3H/HeN and 129svj mice were sensitive to LeTx-induced killing,
whereas macrophages from C57BL/6J mice were resistant. The maximum cell death of sensitive cells was reached 3-4 h after the cells were treated with LeTx (500 ng/ml PA + 500 ng/ml LF), whereas most of the
resistant macrophages were still viable after 16 h of the same
treatment (Fig. 1B). The cytolytic dose of LF for the
macrophages from C3H/HeN (Fig. 1C) and 129svj (data not
shown) mice was ~10 ng/ml when a saturated PA (500 ng/ml) was
present. The resistance of C57BL/6J macrophages to LeTx-induced
cytolysis was not dependent on the dose of LF used in the experiments
because up to a 50-fold increase in LF concentration still could not
produce more cytolysis (Fig. 1C). The cell viability was
measured using crystal violet uptake of live cells and propidium iodide
exclusion (data not shown) with comparable results.
It is known that LF cleaves certain intracellular proteins and that
proteolysis may be essential for cell death. Recent reports have shown
that the cleavage of MEK2 and MKK3 also occurs in LeTx-resistant macrophages (16, 17). This suggests that the resistance to LeTx-induced
cytolysis in resistant macrophages is not due to a defect in
proteolysis mediated by LF. We tested the cleavage of MEK1 in C57BL/6J
and C3H/HeN macrophages. MEK1 was cleaved in the macrophages from both
strains when treated with LeTx (Fig. 1D).
It is known that LeTx-resistant macrophages are not resistant to other
toxins (5). We further tested whether LeTx-resistant macrophages are
generally resistant to death stimuli. Macrophages from C3H/HeN, 129svj,
and C57BL/6J mice were treated with lipopolysaccharides (LPS) plus
IFN- Cellular Activation Can Sensitize Resistant Macrophages
to LeTx-induced Cell Death--
Available data strongly support the
role of macrophage cytolysis in eliciting shock and death in infected
animals (1). Because the mice whose macrophages were resistant to
LeTx-induced cytolysis in vitro still died after infection,
we decided to test whether macrophage activation by bacterial
components, which should happen during bacterial infection, has any
effect on the viability of LeTx-treated resistant macrophages.
Macrophages isolated from C57BL/6J mice (LeTx-resistant macrophages)
were used in the experiments. The cells were treated with LeTx (PA + LF) in the presence or absence of poly-D-glutamic acid
(PGA; 100 µg/ml), the major component of the B. anthracis
capsule (24); peptidoglycan (PG; 10 µg/ml), the cell wall component
of Gram-positive bacteria; or LPS (10 ng/ml), cell wall components of
Gram-negative bacteria. We also treated the cells with retrovirus
(Moloney murine leukemia virus, ~106
plaque-forming units/ml) or zymosan A (100 µg/ml) for comparison. Cell viability was measured 16 h later and is shown in Fig.
2A. The presence of PGA
promoted LeTx-induced C57BL/6J macrophage death. LPS and PG also
significantly enhanced LeTx-induced cell death. Retrovirus and zymosan
A slightly enhanced cell death. The results of an experiment with more
controls is shown in Fig. 2B. LPS, PG, or PGA alone did not
cause cell death. LPS, PG, or PGA combined with PA or LF did not cause
cell death. The cell death of resistant macrophages started at ~5 h
of treatment and reached a maximum at ~16 h (Fig. 2C).
B. anthracis is a Gram-positive bacteria whose capsule
should interact with macrophages, and its cell wall component (PG) can
activate macrophages if the capsule is removed. Thus, the death of
LeTx-treated resistant macrophages can be promoted by cellular
activation with bacterial capsule or cell wall components in
vivo.
Phenotype of LeTx-induced Death of Activated
Macrophages--
LeTx-induced cytolysis of macrophages is more like
necrosis than apoptosis (13, 25, 31), although treatment of macrophages with sublytic amounts of LeTx can trigger some intracellular events of
apoptosis (26, 27). To determine whether C3H/HeN and C57BL/6J macrophages treated with LPS plus a cytolytic dose of LeTx have the
phenotype of apoptosis, three apoptotic features were examined. Nuclear
labeling with Hoechst 33258 is a commonly used method to
detect chromatin condensation, a feature of apoptosis. Macrophages from
C3H/HeN and C57BL/6J mice were left untreated (control) or were
treated with LPS plus LeTx and stained with Hoechst 33258. Chromatin
condensation was not seen throughout the course of cell death of both
C3H/HeN and C57BL/6J macrophages (Fig.
3A and data not shown). As a
positive control, chromatin condensation was observed when C57BL/6J or
C3H/HeN macrophages were treated with LPS plus INF- TNF Plays a Role in Sensitizing LeTx-resistant
Macrophages--
Activated macrophages secrete a number of cytokines,
including TNF, IL-1
We measured TNF production in C57BL/6J macrophages after stimulation
with LPS, PG, and PGA. LPS stimulation led to the highest production of
TNF (Fig. 4B). The PG-stimulated TNF production was about
half of the LPS-induced TNF production, and PGA was less potent in
inducing TNF production. The treatment of cells with LeTx reduced (but
did not abolish) TNF production. The level of TNF production (Fig.
4B) correlated with the death of LeTx-treated resistant
macrophages promoted by these bacterial components (Fig. 2A), suggesting that TNF is a mediator of bacterial
component-promoted death of LeTx-treated cells.
To determine whether the sensitization of LeTx-resistant macrophages to
LeTx-induced death by bacterial components is mediated by TNF, we used
a neutralizing antibody to block TNF. As shown in Fig. 4C,
~50% inhibition of the TNF/LeTx-induced cell death was observed when
anti-TNF antiserum was included in the cell culture medium. Pre-bled
serum had no effect, indicating that the antibody can block the
function of TNF. This antibody also inhibited LPS/LeTx, PG/LeTx, or
PGA/LeTx-induced cell death, supporting the idea that TNF is at least
one of the factors responsible for sensitizing the resistant
macrophages to LeTx-induced cytolysis. Thus, the autocrine/paracrine
effect of TNF plays a role in LeTx-resistant macrophage death.
The bioactivity of TNF is mediated by two TNF receptors,
TNF-RI and TNF-RII. Although human TNF and murine TNF are very
homologous, human TNF binds only to murine TNF-RI (28). We treated
C57BL/6J macrophages with human TNF plus LeTx and found that, in
contrast to murine TNF, human TNF cannot sensitize LeTx-resistant
macrophages to LeTx-induced cell death (Fig. 4D), suggesting
that TNF-RII-mediated signaling is required for sensitizing LeTx-
resistant macrophages.
C57/10ScCr is a mouse strain that has a deletion of the
toll-like receptor-4 (TLR4) gene and will not respond to LPS (29). A
comparable strain with the wild-type TLR4 gene is C57/ScSn. The
macrophages isolated from these two strains of mice were resistant to
LeTx (Fig. 4E). Treatment of LPS in the presence of LeTx led to cell death in C57/ScSn macrophages, but not in C57/10ScCr
macrophages, confirming that TLR4 signaling is required for
LPS-promoted death of LeTx-treated cells. In contrast, TNF plus LeTx
caused cell death in both macrophages, which is consistent with the
idea that bacterial components promote the death of LeTx-treated cells
through the induction of TNF.
TNF can induce the death of a number of different cells.
Although TNF alone had no effect on the viability of macrophages, it is
possible that intracellular signaling activated by TNF can augment the
death of C3H/HeN macrophages induced by LeTx. To examine this
possibility, we tested whether TNF can promote the death of macrophages
treated with sublytic doses of LF. Macrophages from C3H/HeN and
C57BL/6J mice were treated with 500 ng/ml PA and different doses of LF
in the presence or absence of TNF. The levels of cytolysis of cells
were analyzed. As shown in Fig.
5A, TNF stimulation did not
enhance the cell death of C3H/HeN macrophages treated with sublytic or
cytolytic doses of LF. In contrast, TNF increased the cell death of
C57BL/6J macrophages treated with cytolytic doses (Fig. 5B).
The sensitization of LeTx-induced cell death by TNF appears to be a
unique feature of LeTx- resistant macrophages.
TNF/LeTx-induced Cell Death in
LeTx-resistant Macrophages Requires the Proteolytic Activity of LF and
mTor Signaling--
LF(E687C), an inactive LF mutant, has been shown
to be unable to mediate the proteolysis of proteins in vitro
and in cells. Also, LF(E687C) is incapable of causing cytolysis of
LeTx-sensitive macrophages (15). To determine whether the protease
activity of LF is required for TNF/LeTx-induced death of resistant
macrophages, we compared the effect of wild-type LF and LF(E687C) on
the cell viability of resistant macrophages in the presence of TNF
(Fig. 6A). No cell death was
observed in LF(E687C)-treated cells, indicating that the protease
activity of LF is required for TNF/LeTx-induced death of resistant
macrophages.
Caspases have been implicated to play an important role in TNF-induced
cell death of many different cells (30). To test whether caspase is
involved in TNF/LeTx-induced death of LeTx-resistant macrophages, we
used a general caspase inhibitor, Z-VAD-fmk. As shown in Fig.
6B, Z-VAD-fmk itself did not have an effect on the viability
of resistant macrophages, nor did Z-VAD-fmk together with LeTx have any
effect on cell viability. Therefore, caspases are not required for
TNF/LeTx-induced death of LeTx-resistant macrophages.
TNF activates a number of signaling pathways that are involved in cell
survival or the death of a number of different cells (32-35). To
understand how TNF sensitizes LeTx-resistant macrophages, we initiated
our investigation on whether the known signaling pathways that are
activated by TNF are involved in sensitizing LeTx-resistant
macrophages. We used available inhibitors to inhibit the signaling
pathways activated by TNF. Inhibitors (U0126, c-Jun N-terminal kinase
(JNK) inhibitor-2, and SB203580) of three MAPK pathways (extracellular
signal-regulated kinase (ERK), JNK, and p38) did not have any
inhibitory effect on TNF/LeTx-induced cell death, but rather enhanced
cell death (Fig. 6C). The other inhibitors that enhanced
cell death were the protein kinase C inhibitor bisindolylmaleimide II,
the tyrosine kinase inhibitors genistein and herbimycin, and the
phosphatidylinositol 3-kinase inhibitor wortmannin. Because an
oxidative burst was implicated to be responsible for LeTx-induced cell
death (13), we also tested butylated hydroxyanisole, a scavenger of
free radicals, and found no effect on the death of resistant
macrophages (data not shown). The only inhibitor we tested that
inhibited cell death was rapamycin (Fig. 6C). The maximum
inhibition of TNF/LeTx-induced cell death by rapamycin was reach at 10 nM. The known target of rapamycin is mTor
(mammalian target of
rapamycin; also named FRAP and RAFT1), and no nonspecific effect of rapamycin has been reported at 10 nM. Thus, mTor
is most likely involved in cell death.
It is known that the proteolysis by LF impairs the two MAPK pathways,
the ERK and p38 pathways. It was proposed that disruption of certain
intracellular pathways by LF impairs cell survival mechanisms and thus
promotes cell death (15-17, 27). To test whether disruption of the ERK
and/or p38 pathway by LeTx is involved in TNF/LeTx-induced death of
LeTx-resistant macrophages, we used chemical inhibitors to mimic the
inhibitory effect of LF on the ERK and p38 pathways. Inhibition of the
ERK pathway by U0126 and the p38 pathway by SB203580 either
independently or together did not influence the cell viability of
TNF-treated LeTx-resistant macrophages (Fig. 6D and data not
shown), suggesting that cleavage of MEK1/2 and MKK3/6 by LF either is
not involved in TNF/LeTx-induced cell death or is insufficient to
reduce the cell viability. We further tested inhibitors of other
pathways. We used JNK inhibitor-2 to inhibit the JNK pathway (another
MAPK pathway) and did not find any change in the viability of
TNF-treated LeTx-resistant macrophages (Fig. 6D). Similarly,
inhibition of the NF- We have examined the effect of cellular activation of macrophages
on the cell viability of LeTx-treated LeTx-resistant macrophages. We
found that treatment of macrophages with different bacterial components
made LeTx-resistant murine macrophages susceptible to LeTx-induced cell
death. TNF produced by activated macrophages is a key mediator that
sensitizes LeTx-resistant macrophages to LeTx-induced death. We have
determined that the protease activity of LF and mTor activity in cells
are required for TNF/LeTx-induced resistant macrophage death and that
this type of cell death is caspase-independent. Sensitizing
LeTx-resistant macrophages to LeTx-induced cytolysis suggests that host
responses to anthrax infection participate in the macrophage death
in vivo.
LeTx-induced cytolysis of macrophages plays an important role in the
outcome of anthrax infection. Lethality in mice of different strains
resulting from direct injections of LeTx mimics the in vitro
sensitivity of macrophages to LeTx-induced cell death (5). A
contribution of host responses to the lethality of anthrax infection can also be deduced from the study of Welkos et al. (5).
They showed that C3H/HeN and C3H/HeJ mouse strains are killed by
B. anthracis Vollum 1B strain (wild-type) infection with
similar LD50 values (five to six spores). However, the
LD50 of the non-encapsulated toxin-producing strain Sterne
is significantly different. The LD50 of C3H/HeN mice is
8 × 106 spores, whereas C3H/HeJ mice are completely
resistant to the Sterne strain at the highest dose used (2 × 107 spores). These two strains have similar genetic
backgrounds, except that C3H/HeJ mice have a point mutation in TLR4
that impairs signaling initiated by some bacterial cell wall components
such as LPS. It is possible that capsulated B. anthracis can
escape TLR4 recognition and therefore that the TLR4 mutation does not alter the host responses to anthrax infection. However, "naked" B. anthracis can be detected by TLR4, and the TLR4
deficiency impairs macrophage responses, thereby increasing levels of
LD50. Although wild-type B. anthracis does
escape phagocytosis by macrophages and some other host defense
reactions, certain levels of host response should occur. The capsule
should interact with macrophages, and we have shown here that the major
components of the capsule such as PGA can stimulate macrophages to
produce TNF. Although the capsule may prevent PG from interacting with
the macrophages, this interaction would occur if the capsule was
released or damaged. Because macrophages are producers of TNF, the
local concentration of TNF around macrophages can be high. We suggest
that macrophage-produced TNF may be responsible for cytolysis of
LeTx-resistant macrophages in vivo. This contention is
consistent with two published in vivo studies using
neutralizing antibodies of TNF. When the anti-TNF antibody is
administered to BALB/c mice, there is no protective effect from an
injection of LeTx (14). In contrast, administration of the anti-TNF
antibody delays the death of C57BL/6J mice infected with B. anthracis (36). When the macrophages from BALB/c and C57BL/6J mice
were analyzed for their sensitivity to LeTx in vitro, BALB/c
macrophages were found to be sensitive to LeTx, but C57BL/6J macrophages were not. It is possible that because LeTx can directly trigger cytolysis of macrophages in BALB/c mice, inhibition of TNF does
not help the survival of the mice. Although TNF is involved in the
cytolysis of macrophages in anthrax-infected C57BL/6J mice, blocking
TNF delays death.
Life or death of a cell is determined by a balance between death and
survival pathways. TNF is a pleiotropic cytokine produced mainly by
macrophages. TNF is also recognized by macrophages and regulates the
pattern of gene expression (35). TNF-induced cellular responses are
mediated by either one of the two TNF receptors, TNF-RI (p55) and
TNF-RII (p75) (37). Macrophages express both receptors (33). Because
TNF-RI knockout mice have the same sensitivity to anthrax
infection as wild-type mice (36), the sensitization of LeTx-resistant
macrophages to LeTx-induced death by TNF is most likely through
TNF-RII. This notion is consistent with the observation that
human TNF, which is able to bind only to TNF-RI on murine cells, could
not promote the death of LeTx-resistant macrophages (Fig.
4D). TNF can induce caspase-dependent and
-independent cell death (38-40). It is clear that TNF/LeTx-induced
cell death is independent of caspases because Z-VAD-fmk did not inhibit
cell death (Fig. 6B). Because the mechanism of
caspase-independent cell death by TNF is largely unknown, it is unclear
whether the same method is used in TNF/LeTx-induced macrophage death.
Even though TNF induces cell death in many cells, some cell types such
as macrophages proliferate in response to TNF (33). TNF activates
various kinases of the MAPK family and induces various transcription
factors such as NF- Rapamycin was the only inhibitor we tested that could prevent
TNF/LeTx-induced macrophage death (Fig. 6C). The rapamycin
target mTor is a controller of regulatory metabolic responses (43). The
phosphatidylinositol 3-kinase/Akt pathway can regulate mTor in some
systems (43). It is unlikely that the phosphatidylinositol 3-kinase/Akt
pathway is responsible for mTor regulation in our system because
wortmannin or LY294002 did not have the same effect as rapamycin, but
rather had an opposite effect. Because rapamycin did not inhibit
LeTx-induced cytolysis of LeTx-sensitive macrophages (data not shown),
mTor is most likely involved in TNF signaling in TNF/LeTx-induced death
of LeTx-resistant macrophages. How mTor is regulated and how mTor
functions in TNF/LeTx-induced LeTx-resistant macrophage death are a
subject for further investigation.
LeTx causes the proteolysis of certain cellular proteins by triggering
the cytolysis of LeTx-sensitive macrophages. Because of a difference of
three amino acids in the kinesin-like motor protein kif1C, this
proteolysis cannot cause cell death in LeTx-resistant macrophages.
kif1C was suggested to be involved in retrograde vesicle transport, and
TNF may influence this transport to sensitize LeTx-resistant
macrophages. It is also possible that TNF sensitizes LeTx-resistant
macrophages through a mechanism independent of kif1C. LeTx leads to
proteolysis of certain proteins that may damage some cellular
functions. TNF-induced cellular responses under this condition could be
imbalanced and result in cell death. It was reported that human
macrophages are sensitive to LeTx-induced cytolysis in vitro
(13). However, we have tested human macrophages from two donors and
found that both were resistant to LeTx-induced cell death and could be
sensitized through TNF treatment, as was observed in LeTx-resistant
murine macrophages (data not shown). This result fits with a
predication that human macrophages should be resistant to the effect of
LeTx because the human KIF1C gene has the same amino acid
sequence as C57BL/6J and other resistant strains in the region that
determines LeTx sensitivity (17). It appears that different human
populations exhibit differences in the sensitivity of their macrophages
due to the effect of LeTx. Thus, our study on LeTx-resistant macrophage
death is relevant to anthrax infection in humans and provides valuable
insight into the anthrax pathogenesis.
induced by bacterial components was a key factor that
cooperated with LeTx in inducing LeTx-resistant macrophage death. Tumor
necrosis factor-
/LeTx-induced death of LeTx-resistant macrophages
was dependent on mTor (mammalian target of rapamycin), but independent of caspases. Our
data indicate that host responses to anthrax infection contribute to
cytolysis of LeTx- resistant macrophages.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(TNF) induced by bacterial components
is at least one of the factors that cooperate with LeTx in inducing
macrophage death. Our data suggest that the autocrine/paracrine effect
of TNF plays a key role in LeTx-resistant macrophage death in
vivo.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, IL-6,
IFN-
, and human TNF were from R&D Systems (Minneapolis, MN). Mouse
TNF enzyme-linked immunosorbent assay kits were from R&D Systems. All
chemicals were from Sigma or as indicated.
-glycerophosphate, 0.1 mM
phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin, 10 µg/ml
pepstatin A, 1 µg/ml leupeptin, and 1% Triton X-100 and then
sonicated on ice. Cell extracts were obtained by centrifuging the
homogenate at 13,000 rpm for 10 min. These extracts were
electrophoretically resolved in ready-made 10% SDS-polyacrylamide gels
(Bio-Rad), followed by transfer onto nitrocellulose membranes. Membranes were subsequently blocked with 5% skim milk for 30 min, immunoblotted with antibodies, and developed using an enhanced chemiluminescence detection system (ECL, Amersham Biosciences). Antibodies raised against the N terminus of MEK1 were purchased from
Upstate Biotechnology, Inc.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
LeTx (PA + LF)-sensitive and -resistant
macrophages. A, peritoneal macrophages were isolated
from C3H/HeN, 129svj, and C57BL/6J mice and treated with PA (500 ng/ml)
plus LF (500 ng/ml). Cell viability was measured 3 h after
treatment using crystal violet uptake assay. B, macrophages
from C57BL/6J and C3H/HeN mice were treated with PA (500 ng/ml) plus LF
(500 ng/ml) for different periods of time, and cell viability was
measured. C, macrophages from C57BL/6J and C3H/HeN
mice were treated with PA (500 ng/ml) plus different concentrations of
LF as indicated for 16 h. Cell viability was measured.
D, macrophages from C57BL/6J and C3H/HeN mice were treated
with PA (500 ng/ml) plus LF (500 ng/ml) for different periods of time,
and MEK1 and ERK1/2 proteins were analyzed by immunoblotting using
antibodies raised against the MEK1 N terminus and ERK. E,
macrophages from C3H/HeN, 128svj, and C57BL/6J mice were treated with
LPS (10 ng/ml) plus Z-VAD-fmk (zVAD; 10 µM),
LPS plus IFN- (100 units/ml), sodium nitroprusside (SNP;
500 µM), or etoposide (200 µM). Cell
viability was determined 24 h after the treatments. The results
represent means ± S.E. (n = 3-6).
, LPS plus benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone
(Z-VAD-fmk), sodium nitroprusside, or etoposide. All of these reagents
have been shown to cause macrophage death (22, 23). As shown in Fig.
1E, the macrophages from the three strains were killed
equally by these death stimuli. Thus, there is no general resistance to
death in C57BL/6J macrophages.
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Fig. 2.
Bacterial components promote the death of
LeTx-treated LeTx-resistant macrophages. A, C57BL/6J
(LeTx-resistant) macrophages were treated with PA plus LF (500 ng/ml)
alone (Control) or in the presence of Moloney murine
leukemia virus (~106 plaque-forming units/ml), zymosan A
(100 µg/ml), PG (10 µg/ml), LPS (10 ng/ml), or PGA (100 µg/ml).
Cell viability was assayed 16 h after treatment. B,
C57BL/6J macrophages were left untreated (Control) or were
treated with PA (500 ng/ml), LF (500 ng/ml), or PA plus LF in the
presence of LPS, PG, or PGA. Cell viability was measured 16 h
after treatment. C, C57BL/6J macrophages were treated with
PA (500 ng/ml), LF (500 ng/ml), and LPS (10 ng/ml) for different
periods of times, and cell viability was measured.
(Fig.
3A and data not shown). DNA fragmentation is a typical
feature of apoptosis and was examined in the macrophages treated with
LPS plus LeTx. No DNA ladder was detected in C57BL/6J and C3H/HeN
macrophages treated with LPS plus LeTx (Fig. 3B). Treatment
of C57BL/6J macrophages with LPS plus IFN-
was used as positive
control for DNA fragmentation (Fig. 3B, last
lane). Translocation of phosphatidylserine from the inner part of
the plasma membrane to the outer layer is a common early event in apoptosis. Annexin V staining of phosphatidylserine was observed in
macrophages treated with LPS plus IFN-
, but not in macrophages treated with LPS plus LeTx (Fig. 3C). Thus, LPS/LeTx-induced
death of LeTx-resistant macrophages does not have the apoptotic
phenotype, and LPS stimulation does not change the mode of cell death
induced by cytolytic doses of LeTx.
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Fig. 3.
Phenotype of LeTx-induced death of activated
macrophages. A, C3H/HeN and C57BL/6J
macrophages were left untreated (Control) or were treated
with PA (500 ng/ml), LF (500 ng/ml), and LPS (10 ng/ml). C3H/HeN or
C57BL/6J cells were fixed and stained with Hoechst 33258 at 2 or
12 h after treatment, respectively. As a positive control,
C57BL/6J macrophages were treated with LPS (100 ng/ml) plus INF- (10 units/ml) for 24 h and stained with Hoechst 33258. B,
macrophages from C3H/HeN or C57BL/6J mice were treated with PA and LF
or with PA, LF, and LPS as described for A, but for 5 and
24 h, respectively. The positive control was C57BL/6J macrophages
treated with LPS plus IFN-
for 36 h. Genomic DNA was isolated
from C3H/HeN or C57BL/6J macrophages and analyzed by 2% agarose gel
electrophoresis followed by ethidium bromide straining. C,
macrophages from C3H/HeN and C57BL/6J mice were treated as described
for A. The cells were stained with propidium iodide and
annexin V. The percentage of propidium iodide-negative and annexin
V-positive cells was determined by fluorescence-activated cell sorter
analysis (n = 2-3).
, IL-6, and INF-
. We treated C57BL/6J
macrophages with these cytokines of murine origin in the presence or
absence of LeTx and measured cell viability. As shown in Fig.
4A, these cytokines alone did
not affect the viability of macrophages. Interestingly, dramatic cell
death was induced in LeTx-treated cells in the presence of TNF, but not
the other cytokines tested. Thus, TNF can sensitize LeTx-resistant
macrophages to LeTx-induced cell death.
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Fig. 4.
Sensitizing LeTx-resistant macrophages to
LeTx-induced death by TNF. A, C57BL/6J macrophages were
left untreated (Control) or were treated with murine TNF
(200 pM), IL-1 (10 units/ml), IL-6 (250 units/ml), or
IFN-
(100 units/ml) in the presence or absence of PA (500 ng/ml)
plus LF (500 ng/ml). Cell viability was determined 16 h after
treatment. B, C57BL/6J macrophages were left untreated or
were treated with PG, PGA, or LPS in the presence or absence of PA plus
LF for 16 h. TNF concentrations in culture medium were measured by
enzyme-linked immunosorbent assay. C, C57BL/6J macrophages
were treated with PA, LF, and TNF; PA, LF, and LPS; PA, LF, and PG; or
PA, LF, and PGA in the presence or absence (control (Ctrl))
of anti-TNF antiserum (1:500 dilution) for 16 h. The extent of
cell death was analyzed. The percentage of inhibition of cell death was
calculated by ((cell death in the absence of antibody)
(cell
death in the presence of antibody)/cell death in the absence of
antibody). D, macrophages from C57BL/6J mice were treated
with human TNF (hTNF; 500 pM) or murine TNF
(mTNF) in the presence of PA plus LF. Cell viability was
determined 16 h after treatment. E, macrophages from
C57/ScSn or C57/10ScCr mice were treated with PA and LF; PA, LF, and
LPS; or PA, LF, and murine TNF. Cell viability was determined 16 h
after treatment.
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Fig. 5.
Dose curve of LF in mediating the cell death
of TNF-activated C3H/HeN and C57BL/6J macrophages. C3H/HeN
(A) or C57BL/6J (B) macrophages were treated with
PA (500 ng/ml) and different doses of LF in the presence or absence of
TNF (500 pM) for 16 h. Cell viability was
measured.
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Fig. 6.
TNF/LeTx-induced death of resistant
macrophages requires the protease activity of LF and is independent of
caspases. A, C57BL/6J macrophages were left untreated
(Control) or were treated with PA, LF, LF(E687C), PA plus
LF, or PA plus LF(E687C) in the presence of TNF (500 pM).
Cell viability was measured 16 h after treatment. B,
C57BL/6J macrophages were left untreated or were treated with TNF,
Z-VAD-fmk (zVAD), PA plus LF, and their combinations. Cell
viability was measured 16 h after treatment. C,
C57BL/6J macrophages were treated with TNF, PA, and LF in the presence
of U0162 (10 µM), SB203580 (20 µM), JNK
inhibitor-2 (8 µM), bisindolylmaleimide II (3 µM), genistein (50 µM), herbimycin (100 ng/ml), wortmannin (1 µM), LY294002 (10 µM), or rapamycin (20 nM). Cell viability was
measured 16 h after treatment. D, C57BL/6J macrophages
were treated with or without TNF in the presence of U0162 (10 µM), SB203580 (20 µM), JNK inhibitor-2 (8 µM), NF- B SN50 cell-permeable inhibitor peptide (18 µM), bisindolylmaleimide II (3 µM),
LY294002 (10 µM), and their combinations. Cell viability
was measured 16 h after treatment.
B, protein kinase C, or phosphatidylinositol
3-kinase pathways had no effect on the cell viability of TNF-treated
macrophages (Fig. 6D). We tested whether inhibition of all
three MAPK pathways had an effect on the viability of macrophages
treated with TNF. As shown in Fig. 6D, ~20% cell death
was observed when ERK, p38, and JNK were inhibited at the same time.
These data suggest that simultaneous inhibition of multiple MAPK
pathways may have a role in TNF/LeTx-induced macrophage
death. However, the possible nonspecific effect of these inhibitors in
influencing cell viability cannot be excluded. Inhibition of NF-
B
or protein kinase C and the three MAPK pathways at the same time did
not further enhance the death of TNF-treated C57BL/6J macrophages (Fig.
6D). Inhibition of the phosphatidylinositol 3-kinase pathway
together with the three MAPK pathways further enhanced cell killing
(Fig. 6D). Collectively, these data suggest that impairing
any one of the known pathways is not sufficient for sensitizing
LeTx-resistant macrophages. However, it is possible that disruption of
multiple signaling pathways by LF can impair cell survival mechanisms,
although a conclusion cannot be made due to limited information on the
cellular targets of LF.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B in macrophages (32-35). Activation of MAPK has
been shown to be involved in both apoptosis and cell survival of
macrophages depending on the cell death stimuli (41, 42). Recently,
Park et al. (27) showed that LPS stimulation of
macrophages treated with sublytic doses of LF and saturated PA63 (cleaved active form of PA) results in apoptosis. The
dismantling of the p38 MAPK pathway by sublytic doses of LF was
implicated to be partly responsible for the apoptosis of activated
macrophages (27). We have shown here that inhibition of ERK, p38, JNK,
protein kinase C, tyrosine kinase, and phosphatidylinositol 3-kinase
pathways enhanced TNF/LeTx-induced death of LeTx-resistant macrophages (Fig. 6C), which supports the idea that damage to certain
intracellular signaling pathways by LF may impair cell survival
mechanisms (27). Inhibition of any single pathway did not have an
effect on the viability of TNF-treated macrophages (Fig.
6D), suggesting that disruption of a single signaling
pathway cannot mimic the LF effect on survival mechanisms. This
speculation was supported by the observation that a combination of
inhibitors of MAPKs and phosphatidylinositol 3-kinase significantly
induced cell death in TNF-treated macrophages (Fig. 6D). In
contrast to the reported effect of sublytic doses of LF (27), apoptotic
features could not be found in macrophages treated with cytolytic doses
of LeTx in the presence of LPS stimulation (Fig. 3). It appears that
the phenotype of cell death is determined by the level of LF
damage rather than cellular activation because sublytic doses of
LeTx alone already triggers apoptotic features (26).
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ACKNOWLEDGEMENTS |
---|
We thank Dr. R. J. Collier for helpful discussion and Tonya Thomson for excellent secretarial work.
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FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health Grant AI-41637 and a grant from the California Cancer Research Program (to J. H.). This is Publication 15156-IMM from the Department of Immunology, Scripps Research Institute (La Jolla, CA).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. Section 1734 solely to indicate this fact.
§ Both authors contributed equally to this work.
¶ Supported by a Canadian Institutes of Health Research fellowship.
** To whom correspondence should be addressed: Dept. of Immunology, Scripps Research Inst., 10550 N. Torrey Pines Rd., La Jolla, CA 92037. Tel.: 858-784-8704; Fax: 858-784-8665; E-mail: jhan@scripps.edu.
Published, JBC Papers in Press, December 17, 2002, DOI 10.1074/jbc.M209279200
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ABBREVIATIONS |
---|
The abbreviations used are:
EF, edema factor;
LF, lethal factor;
PA, protective antigen;
LeTx, lethal toxin;
MAPK, mitogen-activated protein kinase;
MKK, mitogen-activated protein kinase
kinase;
TNF, tumor necrosis factor-;
IL, interleukin;
IFN-
, interferon-
;
MEK, mitogen-activated protein kinase/extracellular
signal-regulated kinase kinase;
MOPS, 4-morpholinepropanesulfonic acid;
LPS, lipopolysaccharide(s);
Z-VAD-fmk, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone;
PGA, poly-D-glutamic acid;
PG, peptidoglycan;
TNF-R, tumor
necrosis factor receptor;
TLR4, toll-like receptor-4;
JNK, c-Jun
N-terminal kinase;
ERK, extracellular signal-regulated kinase.
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REFERENCES |
---|
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---|
1. |
Dixon, T. C.,
Meselson, M.,
Guillemin, J.,
and Hanna, P. C.
(1999)
N. Engl. J. Med.
341,
815-826 |
2. |
Sellman, B. R.,
Nassi, S.,
and Collier, R. J.
(2001)
J. Biol. Chem.
276,
8371-8376 |
3. | Leppla, S. H. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 3162-3166[Abstract] |
4. | Klimpel, K. R., Arora, N., and Leppla, S. H. (1994) Mol. Microbiol. 13, 1093-1100[Medline] [Order article via Infotrieve] |
5. | Welkos, S. L., Keener, T. J., and Gibbs, P. H. (1986) Infect. Immun. 51, 795-800[Medline] [Order article via Infotrieve] |
6. | Pezard, C., Berche, P., and Mock, M. (1991) Infect. Immun. 59, 3472-3477[Medline] [Order article via Infotrieve] |
7. | Bradley, K. A., Mogridge, J., Mourez, M., Collier, R. J., and Young, J. A. (2001) Nature 414, 225-229[CrossRef][Medline] [Order article via Infotrieve] |
8. | Wesche, J., Elliott, J. L., Falnes, P. O., Olsnes, S., and Collier, R. J. (1998) Biochemistry 37, 15737-15746[CrossRef][Medline] [Order article via Infotrieve] |
9. |
Molloy, S. S.,
Bresnahan, P. A.,
Leppla, S. H.,
Klimpel, K. R.,
and Thomas, G.
(1992)
J. Biol. Chem.
267,
16396-16402 |
10. | Elliott, J. L., Mogridge, J., and Collier, R. J. (2000) Biochemistry 39, 6706-6713[CrossRef][Medline] [Order article via Infotrieve] |
11. | Milne, J. C., and Collier, R. J. (1993) Mol. Microbiol. 10, 647-653[Medline] [Order article via Infotrieve] |
12. |
Erwin, J. L.,
DaSilva, L. M.,
Bavari, S.,
Little, S. F.,
Friedlander, A. M.,
and Chanh, T. C.
(2001)
Infect. Immun.
69,
1175-1177 |
13. | Hanna, P. C., Kruskal, B. A., Ezekowitz, R. A., Bloom, B. R., and Collier, R. J. (1994) Mol. Med. 1, 7-18[Medline] [Order article via Infotrieve] |
14. | Hanna, P. C., Acosta, D., and Collier, R. J. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 10198-10201[Abstract] |
15. |
Duesbery, N. S.,
Webb, C. P.,
Leppla, S. H.,
Gordon, V. M.,
Klimpel, K. R.,
Copeland, T. D.,
Ahn, N. G.,
Oskarsson, M. K.,
Fukasawa, K.,
Paull, K. D.,
and Vande Woude, G. F.
(1998)
Science
280,
734-737 |
16. | Pellizzari, R., Guidi-Rontani, C., Vitale, G., Mock, M., and Montecucco, C. (1999) FEBS Lett. 462, 199-204[CrossRef][Medline] [Order article via Infotrieve] |
17. | Watters, J. W., Dewar, K., Lehoczky, J., Boyartchuk, V., and Dietrich, W. F. (2001) Curr. Biol. 11, 1503-1511[CrossRef][Medline] [Order article via Infotrieve] |
18. |
Dorner, C.,
Ciossek, T.,
Muller, S.,
Moller, P. H.,
Ullrich, A.,
and Lammers, R.
(1998)
J. Biol. Chem.
273,
20267-20275 |
19. | Ulevitch, R. J., and Tobias, P. S. (1995) Annu. Rev. Immunol. 13, 437-457[CrossRef][Medline] [Order article via Infotrieve] |
20. | Aderem, A., and Ulevitch, R. J. (2000) Nature 406, 782-787[CrossRef][Medline] [Order article via Infotrieve] |
21. | Border, J. R. (1988) Arch. Surg. 123, 285-286[CrossRef][Medline] [Order article via Infotrieve] |
22. |
Kim, S. O.,
Ono, K.,
and Han, J.
(2001)
Am. J. Physiol.
281,
L1095-L1105 |
23. | Kim, S. O., and Han, J. (2001) J. Endotoxin Res. 7, 292-296[Medline] [Order article via Infotrieve] |
24. | Grell, M., Douni, E., Wajant, H., Lohden, M., Clauss, M., Maxeiner, B., Georgopoulos, S., Lesslauer, W., Kollias, G., and Pfizenmaier, K. (1995) Cell 83, 793-802[Medline] [Order article via Infotrieve] |
25. | Lin, C. G., Kao, Y. T., Liu, W. T., Huang, H. H., Chen, K. C., Wang, T. M., and Lin, H. C. (1996) Curr. Microbiol. 33, 224-227[CrossRef][Medline] [Order article via Infotrieve] |
26. | Popov, S. G., Villasmil, R., Bernardi, J., Grene, E., Cardwell, J., Wu, A., Alibek, D., Bailey, C., and Alibek, K. (2002) Biochem. Biophys. Res. Commun. 293, 349-355[CrossRef][Medline] [Order article via Infotrieve] |
27. |
Park, J. M.,
Greten, F. R., Li, Z. W.,
and Karin, M.
(2002)
Science
297,
2048-2051 |
28. |
Ameloot, P.,
Fiers, W., De,
Bleser, P.,
Ware, C. F.,
Vandenabeele, P.,
and Brouckaert, P.
(2001)
J. Biol. Chem.
276,
37426-37430 |
29. |
Poltorak, A., He, X.,
Smirnova, I.,
Liu, M. Y.,
Huffel, C. V., Du, X.,
Birdwell, D.,
Alejos, E.,
Silva, M.,
Galanos, C.,
Freudenberg, M.,
Ricciardi-Castagnoli, P.,
Layton, B.,
and Beutler, B.
(1998)
Science
282,
2085-2088 |
30. | Strasser, A., O'Connor, L., and Dixit, V. M. (2000) Annu. Rev. Biochem. 69, 217-245[CrossRef][Medline] [Order article via Infotrieve] |
31. | Kau, J. H., Lin, C. G., Huang, H. H., Hsu, H. L., Chen, K. C., Wu, Y. P., and Lin, H. C. (2002) Curr. Microbiol. 44, 106-111[Medline] [Order article via Infotrieve] |
32. | Winston, B. W., Chan, E. D., Johnson, G. L., and Riches, D. W. H. (1997) J. Immunol. 159, 4491-4497[Abstract] |
33. |
Mukhopadhyay, A.,
Suttles, J.,
Stout, R. D.,
and Aggarwal, B. B.
(2001)
J. Biol. Chem.
276,
31906-31912 |
34. |
Chan, E. D.,
Winston, B. W.,
Jarpe, M. B.,
Wynes, M. W.,
and Riches, D. W.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
13169-13174 |
35. | Riches, D. W., Chan, E. D., and Winston, B. W. (1996) Immunobiology 195, 477-490[Medline] [Order article via Infotrieve] |
36. | Kalns, J., Scruggs, J., Millenbaugh, N., Vivekananda, J., Shealy, D., Eggers, J., and Kiel, J. (2002) Biochem. Biophys. Res. Commun. 292, 41-44[CrossRef][Medline] [Order article via Infotrieve] |
37. | Smith, C. A., Farrah, T., and Goodwin, R. G. (1994) Cell 76, 959-962[Medline] [Order article via Infotrieve] |
38. | Rathmell, J. C., and Thompson, C. B. (1999) Annu. Rev. Immunol. 17, 781-828[CrossRef][Medline] [Order article via Infotrieve] |
39. |
Vercammen, D.,
Beyaert, R.,
Denecker, G.,
Goossens, V.,
Van Loo, G.,
Declercq, W.,
Grooten, J.,
Fiers, W.,
and Vandenabeele, P.
(1998)
J. Exp. Med.
187,
1477-1485 |
40. | Holler, N., Zaru, R., Micheau, O., Thome, M., Attinger, A., Valitutti, S., Bodmer, J. L., Schneider, P., Seed, B., and Tschopp, J. (2000) Nat. Immunol. 1, 489-495[CrossRef][Medline] [Order article via Infotrieve] |
41. |
Mohr, S.,
McCormick, T. S.,
and Lapetina, E. G.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
5045-5050 |
42. |
Petrache, I.,
Choi, M. E.,
Otterbein, L. E.,
Chin, B. Y.,
Mantell, L. L.,
Horowitz, S.,
and Choi, A. M.
(1999)
Am. J. Physiol.
277,
L589-L595 |
43. | Schmelzle, T., and Hall, M. N. (2000) Cell 103, 253-262[Medline] [Order article via Infotrieve] |