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J Biol Chem, Vol. 273, Issue 49, 32895-32900, December 4, 1998
From the Skirball Institute, Department of Microbiology and Kaplan
Cancer Center, New York University School of Medicine, New York, New
York 10016, the § Unité de Pathogénie
Microbienne Moléculaire, INSERM U389, Institut Pasteur, 28 Rue du
Docteur Roux, 75724 Paris Cedex 15, France, ¶ BASF Bioresearch
Corporation, Worcester, Massachusetts 01605-4314, the We report here that the Shigella invasion plasmid
antigen (Ipa)B, which is sufficient to induce apoptosis in macrophages, binds to caspase (Casp)-1, but not to Casp-2 or Casp-3. Casp-1 is
activated and its specific substrate interleukin-1 Shigellae are the etiological agents of bacillary dysentery, an
acute form of diarrhea accompanied by blood and mucus. Dysentery is an
epidemiologically important disease which is often fatal in children.
Shigellae are invasive bacteria that penetrate the colonic tissue and
initiate an acute inflammation which is predominantly responsible for
the ensuing symptoms (1, 2). In vitro, Shigellae invade
eukaryotic cells and escape the phagosome to reach the cytoplasm (3).
Both invasion and phagosome escape require the secretion of the three
invasion plasmid antigens
(Ipa)1 proteins:
IpaB, IpaC, and IpaD (4, 5).
Shigella induces apoptosis in macrophages, but not other cell types
(6). In vivo, induction of apoptosis in Shigellosis was
demonstrated in an animal model (7) and dysenteric patients (8). IpaB
is sufficient to induce apoptosis in macrophages (9). This invasin is
secreted by intracellular bacteria and distributed in the cell
cytoplasm (10) where it binds to a caspase, and activates apoptosis
(9).
Caspases are cysteine proteases that cleave after aspartate residues
and activate apoptosis in eukaryotic cells. Eleven different caspases
have been identified in mice and humans, each with a distinct substrate
specificity. These enzymes are translated as zymogens which
autocatalyze their cleavage into mature enzymes. The amino-terminal
domain of caspase precursors does not contribute to the mature enzyme
and may be involved in the regulation of maturation. Mature caspases
are heterodimers formed by two subunits derived from the
carboxyl-terminal domain of the precursor (11).
Based on sequence comparison and substrate specificity, caspases can be
classified into three subfamilies. Caspase-1 (Casp-1, interleukin-1 Casp-11 (Ich-3) is closely related to Casp-1 (20). As opposed to Casp-1
which is constitutively expressed, Casp-11 is induced in macrophages
only after LPS stimulation. Overexpression of Casp-11 results in
IL-1 Casp-3, the mammalian caspase most highly related to Ced-3, cleaves a
number of homeostatic, repair, and structural proteins (22-24). Casp-3
is activated in many different systems. In contrast to
casp-1- and casp-11-deficient mice,
casp-3 knockouts die prematurely and have a profound defect
in brain development (25). Thymocytes from these mice are susceptible,
however, to some apoptotic stimuli. Casp-3 is therefore required in
many, but not all pathways that lead to programmed cell death. Casp-2
is alternatively spliced and generates a pro- and an anti-apoptotic
molecule (26). In casp-2 Here, we show that IpaB binds to Casp-1, but not Casp-2 or Casp-3. As
expected, Casp-1 is activated and IL-1 Ligand Blot of His-tagged Caspase Fusion
Proteins--
His-tagged Casp-1 was constructed by releasing the
cDNA from pJ.348 (17) by a BamHI, SalI
digestion, cloning into pGSTag (30), and subsequently subcloning into
pRSETA (Invitrogen, Carlsbad, CA). casp-2 (31) and
casp-3 (22) were amplified by polymerase chain reaction from
pMSN2.4 and pSK-CPP32
The constructs (His-casp-1, -2, and-3)
were transformed into Escherichia coli BL21(DE3)/pLysS and
induced with 0.5 mM
isopropyl-1-thio-
The probes for the ligand blot experiment, GSTag-IpaB and GSTag, were
purified and labeled with protein kinase A/[ Bacteria and Growth Conditions--
M90T, an invasive isolate of
Shigella flexneri serotype 5, is our virulent strain of
reference (32). An IpaB deletion mutant of M90T, referred to as
Collection of Peritoneal Macrophages--
casp-1 (18),
casp-3 (25), and casp-11 (21) knock-out mice
between 6 and 7 weeks old were obtained from the Animal Resource Facility at BASF Bioresearch Corp., Yale and Harvard Universities, respectively. p53 knock-out and C57BL/6 control mice were
obtained from Jackson Laboratory (Bar Harbor, ME). Four days after an
intraperitoneal injection of 1 ml of thioglycolate, macrophages were
collected by peritoneal lavage with cold phosphate-buffered saline and
washed. Macrophages were isolated by adherence at 37 °C for 90 min
and cultured in RPMI-1640 glutamine medium (Life Technologies, Inc.) supplemented with 10% fetal calf serum (Life Technologies, Inc.), penicillin (10,000 units/ml), and streptomycin (10,000 µg/ml).
Macrophage Infection and Cytotoxicity Assays--
Macrophages
were infected at a multiplicity of infection of 100 bacteria (colony
forming units) per cell as described previously (33). Cytotoxicity was
measured using the CytoTox 96 assay, according to the manufacturer's
instructions (Promega, Madison, WI). LDH release is a measure of cell
death, not specifically of apoptosis. Nevertheless, we have previously
established that Shigella kills by apoptosis (6). Furthermore, in order
to detect apoptotic cells, similar preparations were fixed with 4%
paraformaldehyde, permeabilized with 0.5% Triton X-100, stained with
propidium iodide at a concentration of 0.01 mg/ml (Molecular Probes,
Inc., Eugene, OR), and observed with a fluorescence microscope.
Macrophage like RAW cells transfected with bcl-2 were kindly
provided by Dr. Brune (34). For Etoposide (Sigma) experiments,
106 cells were treated with 50 µM for 24 h.
Maturation of ICE and IL-1 IpaB Binds to Casp-1, But Not to Casp-2 or Casp-3--
The
S. flexneri invasin IpaB was shown to bind to a caspase in
macrophage lysates both in vitro and in vivo (9).
The IpaB-binding caspase was tentatively identified as Casp-1 using a
polyclonal antiserum (9). To confirm that Casp-1 binds IpaB, and test whether other caspases also bind this bacterial protein, we performed a
ligand blot assay. We chose to test Casp-1, Casp-2, and Casp-3 as
representative members of the three caspase subfamilies (11).
Purified His-tagged Casp-1, Casp-2, and Casp-3 were resolved by
SDS-polyacrylamide gel electrophoresis and transferred onto nitrocellulose. An anti-T7 monoclonal antibody which recognizes an
epitope within the His-tag was used to verify the amount of protein
loaded into each lane (Fig. 1). We used
protein-kinase A/[ Casp-1 Is Activated during Shigella Infections--
Since IpaB
binds to Casp-1 we tested whether Casp-1 matures from its zymogen to
its active form during infection. In these experiments we infected
macrophages activated with LPS, to be able to detect IL-1
IL-1 Casp-1 Casp-3 and Casp-11 Are Not Necessary for Shigella-induced
Apoptosis--
We tested whether macrophages from casp-3
knock-out mice (25) were susceptible to Shigella-induced cytotoxicity.
Peritoneal macrophages isolated from wild type and casp-3
Similar results were obtained with macrophages from casp-11
knock-out mice (21). Casp-11 appears to be an upstream regulator of
Casp-1 activation (21). As shown in Fig. 4B, virulent
Shigella killed macrophages isolated from control or casp 11 Shigella-induced Apoptosis Is Independent of p53 and Cannot be
Inhibited by Bcl-2--
To further investigate the nature of the
apoptotic cascade engaged in macrophages infected by Shigella, we
tested the role of the tumor suppressor, p53, as well as the
apoptosis inhibitor oncogene bcl-2. p53 is necessary for
apoptosis induced by numerous stimuli (36). Conversely, apoptosis is
often inhibited by overexpression of Bcl-2 (37, 38). Shigella killed
p53
In order to test whether overexpression of Bcl-2 affects
Shigella-induced apoptosis, the macrophage-like cell line RAW stably transfected with either the vector alone, or the vector expressing Bcl-2 (34) was infected with M90T or BS176. As shown in Fig. 5B, overexpression of Bcl-2 did not protect macrophages from
Shigella killing. BS176 is not cytotoxic to either bcl-2 or
vector-transfected cells. Similar results were obtained with
differentiated U937 cells overexpressing Bcl-xL, another
anti-apoptotic protein of the Bcl-2 family (Ref. 39, data not shown).
Expression of Bcl-2 in the transfected macrophages was confirmed by
Western blot (data not shown). Furthermore, in order to determine
whether the Bcl-2 overexpressed in RAW cells was functional, we treated
these macrophages with the topoisomerase inhibitor Etoposide, an
inducer of apoptosis that is known to be inhibited by Bcl-2.
Twenty-four hours after treatment almost 50% of the vector control
cells yet only 5% of the Bcl-2 expressing cells died (Fig.
5C), indicating that Bcl-2 expressed in RAW cells is biologically active.
Among the caspases, Casp-1 appears to be unique since it induces
apoptosis and activates two potent proinflammatory cytokines, IL-1 Although Casp-1 induces apoptosis when overexpressed in tissue culture
cells (17) the role of this enzyme in apoptosis has remained enigmatic.
casp-1 The Shigella invasin IpaB binds specifically to Casp-1 but not Casp-2
or Casp-3 (Fig. 1). Based on sequence homology, Casp-1, Casp-2, and
Casp-3 are representatives of the three caspase subfamilies (11). It
seems unlikely that IpaB binds to caspases closely related to Casp-1,
since Shigella kills macrophages derived from casp-11 Casp-1 is processed shortly after Shigella infection suggesting that
IpaB can promote this process (Fig. 2A). Cleavage of pro-IL-1 Casp-1 is necessary for Shigella-induced apoptosis since these bacteria
do not kill macrophages from mice with a targeted deletion in this
protease (Fig. 3). These results are surprising, as macrophages
isolated from casp-1 Recently, Casp-11 was shown to regulate Casp-1 activation (21). Data
presented here suggest that Shigella directly activates apoptosis
through Casp-1, since macrophages from casp-11 The pro-apoptotic function of Casp-1 in Shigella-induced apoptosis
appears to be independent of mature IL-1 Consistent with the model that Shigella induces apoptosis by directly
activating Casp-1 is the observation that this process does not require
p53 (Fig. 5). p53 is a transcriptional activator that is necessary for
apoptosis initiated by diverse stimuli after G1 arrest in
the cell cycle (36). The susceptibility of p53 We propose the following model for apoptosis during Shigellosis.
Shigella secretes IpaB into the cytoplasm (10) where it binds to
Casp-1. Casp-1 is then activated and cleaves pro-IL-1 Macrophages infected with Shigella release large amounts of IL-1 (33)
and treatment of animals with the IL-1 receptor antagonist prevents
inflammation after infection with Shigella (44). Here we demonstrate
that the Shigella invasin IpaB specifically binds to Casp-1 and that
this caspase is absolutely required for Shigella-induced apoptosis.
These data indicate that in contrast to developmental or homeostatic
apoptosis, Shigella-induced apoptosis requires Casp-1. In view of the
dual role of Casp-1 as a pro-apoptotic and pro-inflammatory enzyme, we
propose that Casp-1-dependent cell death results in inflammation. Since
IL-1 Dysentery is self-limiting if patients do not succumb to dehydration.
Thus, although the inflammatory response initially allows the disease
to progress, it eventually controls the infection. It is interesting to
speculate that Casp-1, with its dual pro-apoptotic and pro-inflammatory
function, evolved together with IL-1 and IL-18 as triggers of an acute
inflammatory response.
We thank Dr. Doug Miller for the anti-Casp-1
antibody, Dr. Craig B. Thompson for the Bcl-xL transfected
cells, Dr. B. Brune for the Bcl-2 expressing macrophages, and
Antonios O. Aliprantis for careful reading and helpful comments.
*
This work was supported by a collaborative NATO Grant CRG
960044 (shared by P. J. S. and A. Z.) and National
Institutes of Health Grants AI37720 and AI42780 (to A. Z.).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.
**
Investigator of the Howard Hughes Medical Institute.
§§
To whom correspondence should be addressed: Skirball Institute,
New York University Medical Center, 540 First Ave., New York, NY 10016. Tel.: 212-263-7058; Fax: 212-263-5711; E-mail:
zychlinsky{at}saturn.med.nyu.edu.
The abbreviations used are:
Ipa, invasion
plasmid antigen; Casp, caspase; IL, interleukin; LPS, lipopolysaccharide.
2
H. Hilbi, J. E. Moss, D. Hersh, Y. Chen, J. Arondel, S. Banerjee, R. A. Flavell, J. Yuan, P. J. Sansonetti, and A. Zychlinsky, unpublished results.
3
H. Hilbi, J. E. Moss, D. Hersh, Y. Chen, J. Arondel, S. Banerjee, R. A. Flavell, J. Yuan, P. J. Sansonetti, and A. Zychlinsky, unpublished observation.
4
D. Hersh, D. M. Monack, M. R. Smith, N. Ghori,
S. Falkow, and A. Zychlinsky, manuscript in preparation.
Shigella-induced Apoptosis Is Dependent on Caspase-1 Which
Binds to IpaB*
,
**,
, Section of
Immunobiology, Howard Hughes Medical Institute, Yale University School
of Medicine, New Haven, Connecticut 06510, and the
Department of Cell Biology, Harvard Medical
School, Boston, Massachusetts 02115
ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References
is cleaved shortly after Shigella infection. Macrophages isolated from Casp-1 knock-out mice are not susceptible to Shigella-induced apoptosis, although they respond normally to other apoptotic stimuli. Shigella kills macrophages from casp-3, casp-11, and
p53 knock-out mice as well as macrophages overexpressing
Bcl-2. We propose that Shigella induces apoptosis by directly
activating Casp-1 through IpaB, bypassing signal transduction events
and caspases upstream of Casp-1. Taken together these data indicate
that Shigella-induced apoptosis is distinct from other forms of
apoptosis and seems uniquely dependent on Casp-1.
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
converting enzyme), Casp-2 (Nedd-2, Ich-1), and Casp-3
(CPP32/Yama/Apopain) are prototypic examples of each subfamily (11,
12). Casp-1 was originally purified based on its ability to convert the
proinflammatory cytokine interleukin-1 (IL-1), from a 30-kDa
precursor to a biologically active 17-kDa protein (13, 14). Casp-1 also
activates IL-18, a cytokine that induces interferon-
(15, 16).
Caspases are homologues of the Caenorhabditis elegans cell
death gene ced-3. Casp-1, as well as most other caspases, activates apoptosis when overexpressed in culture cell lines (17). Surprisingly, mice with a targeted deletion in casp-1 do not
have an overt apoptosis-related phenotype. Yet, they are relatively resistant to endotoxin-induced shock, presumably because they cannot
process IL-1 (18, 19) and IL-18 (15, 16). ATP induced apoptosis is as
efficient in macrophages explanted from casp-1 
/ as in
macrophages from wild type mice (18). Thus, Casp-1 is not believed to
be necessary for apoptosis during development or in homeostasis.
cleavage, even though Casp-11 does not directly cleave this
cytokine. Moreover, overexpression of Casp-1 in cells from
casp-11 / mice does not induce apoptosis.
Similar to casp-1 / mice, casp-11 knock-outs
develop normally and are resistant to endotoxin-induced shock (21).
Taken together, these data suggest that Casp-11 acts upstream of
Casp-1.
/ mice apoptosis mediated by
perforin and granzyme B was defective but developmental cell death of
motor neurons was accelerated (27). The position of Casp-2 in a
"caspase cascade" is still unclear (28, 29).
is cleaved during a Shigella
infection. Using macrophages elicited from mice with different targeted
deletions, we demonstrate that Shigella requires Casp-1, but not Casp-3
or Casp-11 to induce apoptosis. Shigella-induced apoptosis proceeds in
the absence of the apoptosis promoter p53 and in the presence of the
anti-apoptotic protein Bcl-2. We propose a model where Shigella
bypasses upstream apoptotic regulators to directly activate Casp-1.
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
, respectively (kindly provided by Dr. S. Kumar; Hanson Center for Cancer Research, Adelaide, Australia, and Dr.
E. S. Alnemri; Jefferson Cancer Institute, Philadelphia, PA). The
polymerase chain reaction products were cloned into the
BamHI and PstI sites of pRSETA and sequenced.
-d-galactopyranoside at an OD595 of
0.8-1.0 (1 h at 37 °C). Bacterial pellets were resuspended in
binding buffer containing 1 mM phenylmethylsulfonyl fluoride, lysed by sonication, and the His-tagged caspases were purified by nickel chelate chromatography according to the
manufacturer's instructions (Novagen, Madison, WI). His-Casp-3 was
soluble and stable under the above conditions, while His-Casp-2 had to
be solubilized from inclusion bodies by addition of 1.5% Sarcosyl. His-Casp-1 and -2 were stabilized by addition of either 100 µM acetyl-Tyr-Val-Ala-Asp-CHO (YVAD-CHO, Biomol, Plymouth
Metting, PA) or 100 µM YVAD-CHO and
acetyl-Asp-Glu-Val-Asp-CHO (DEVD-CHO), respectively. The purified
His-tagged caspases were quantified by Western blot using a monoclonal
anti-T7-Tag antibody (Novagen).
-32P]ATP
as described (9). Nitrocellulose membranes containing similar amounts
of His-Casp-1, -2, and 3 were blocked for 2 h at room temperature
in 20 mM Tris, pH 7.5, 20 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol, 0.5% Triton
X-100, 5% milk. The blocked membranes were probed with
32P-labeled GSTag-IpaB ([32P]GSTag-IpaB) or
[32P]GSTag, diluted in blocking buffer containing 50 mM NaCl but no milk (1.5 h, room temperature), and washed
twice for 30 min with blocking buffer without milk. The radiolabeled
membranes were exposed to a PhosphorImager screen.
ipaB (4), or a plasmid-less non-virulent derivative,
BS176, were used as negative controls. Bacteria were grown in tryptic
soy broth at 37 °C with aeration. The bacteria were washed and
resuspended in RPMI 1640 medium before infection of macrophages.
in Peritoneal Macrophages Infected
with Shigella--
Peritoneal macrophages were elicited from Swiss
Webster mice, selected and infected as described above. 0.5 × 106 macrophages were activated overnight with 1 µg/ml LPS
(Shigella serotype 1A; Sigma), when indicated. Prior to infection, the
cells were washed with serum-free RPMI 1640, and, when indicated, the cells were incubated for 1 h with 50 µM YVAD-CMK.
After infection at a multiplicity of infection of 50 the macrophages
were lysed in situ at given time points as described
previously (35). Samples containing equal protein amounts were
separated on 15% SDS-polyacrylamide gels, transferred to
nitrocellulose, and analyzed by Western blot using either a rabbit
anti-Casp-1 (kindly provided by Dr. D. Miller; Merck, Rahway, NJ), or a
goat anti-IL-1 (R&D Systems, Minneapolis, MN) antibody.
RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-32P]ATP to radiolabel purified
GSTag and GSTagIpaB (30). Identical membranes containing the different
caspases were probed with either [32P]GSTagIpaB or
[32P]GSTag as a control. As shown in Fig. 1,
[32P]GSTagIpaB binds to Casp-1 but not to Casp-2 or
Casp-3, while [32P]GSTag did not bind to any of these
caspases. IpaB bound both to the Casp-1 zymogen of 45 kDa as well as to
a degradation product of around 30 kDa. This indicates that IpaB
specifically binds to Casp-1.
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Fig. 1.
IpaB binds to Casp-1 but not to Casp-2 or
Casp-3. Purified His-tagged Casp-1 (lane 1), Casp-2
(lane 2), and Casp-3 (lane 3) were resolved by
SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose,
and probed with an anti-T7 Tag antibody to assess the amount of protein
loaded. Identical membranes were probed with either
[32P]GSTagIpaB or [32P]GSTag as a control.
[32P]GSTagIpaB bound to Casp-1 but not to the other two
caspases. The control [32P]GSTag did not bind to any of
the caspases.
. Lysates
were made at the indicated time points and analyzed by immunoblots for
the maturation of Casp-1 and IL-1. We detected Casp-1 maturation
from the 45-kDa precursor to the 20-kDa subunit as early as 20 min
after infection (Fig. 2A).
This anti-Casp-1 antibody recognizes the 20 kDa, but not the 10-kDa
subunit of the mature enzyme. As expected, expression of Casp-1 was not
up-regulated after activation with LPS and Casp-1 did not mature in
macrophages infected with the non-virulent strain BS176. We also tested
whether the caspase inhibitor YVAD could block caspase maturation. YVAD
inhibited Casp-1 maturation (Fig. 2B), as well as
Shigella-induced apoptosis (9).
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Fig. 2.
Casp-1 matures during S. flexneri
infection. Naive or LPS-activated peritoneal macrophages
from wild type mice were infected with virulent (M90T) or
avirulent (BS176) S. flexneri. Lysates collected
at the indicated post-infection time points were analyzed by Western
blot. A, Western probed with an anti-Casp-1 antibody. Casp-1
is expressed at comparable levels in naive and LPS-activated
macrophages and matures shortly after infection with M90T as indicated
by the appearance of the 20-kDa band. B, Western probed with
an anti-Casp-1 antibody of macrophages treated with the Casp-1
inhibitor YVAD-cmk before infection. Casp-1 maturation is blocked by
YVAD-cmk. C, Western probed with an anti-IL-1 antibody.
IL-1 is expressed only in LPS-activated macrophages and matures
after infection with M90T as indicated by the appearance of the 17-kDa
band. D, Western probed with an anti-IL-1 antibody of
macrophages treated with the Casp-1 inhibitor YVAD-cmk before
infection. As expected, IL-1 maturation is blocked by
YVAD-cmk.
, a substrate of Casp-1, was cleaved with similar kinetics to
Casp-1. Not surprisingly, expression of IL-1 was only detected in
LPS-activated macrophages. IL-1 was not cleaved in macrophages
infected with BS176 (Fig. 2C). IL-1 maturation was significantly retarded in macrophages preincubated with YVAD (Fig. 2D). These data show that Casp-1 is activated in
Shigella-infected macrophages.
/ Macrophages Are Resistant to Shigella-induced
Apoptosis in Vitro--
To further investigate the role of Casp-1 in
Shigella-induced apoptosis, we infected peritoneal macrophages from
wild type and casp-1 / mice (18), with either
the S. flexneri wild type strain M90T or an isogenic strain
with a deletion in ipaB (ipaB, Ref. 4). M90T
killed more than 90% of a culture of macrophages from wild type mice
within 3 h of infection (Fig. 3). In
contrast, macrophages from casp-1 / mice were
resistant to Shigella-induced cell death. Macrophages infected with
ipaB were not killed. Infected macrophages were also
stained with propidium iodide and scored for apoptotic morphology.
These experiments confirmed that S. flexneri induces
apoptosis in wild type but not in casp-1 / macrophages (data not shown). Thus, unlike apoptosis induced by other
stimuli (18), Shigella-mediated macrophage death requires casp-1.
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Fig. 3.
Macrophages from casp-1 /
mice are not susceptible to S. flexneri induced
apoptosis. Peritoneal macrophages were isolated from wild type or
casp-1 / mice and infected with either M90T or a
deletion mutant in ipaB (ipaB). At the
indicated time points, cytotoxicity was determined by measuring the
release of cytoplasmic lactate dehydrogenase (LDH). Only
macrophages from wild type mice are susceptible to Shigella-induced
apoptosis.
/ mice were killed with equal efficiency by Shigella
(Fig. 4A). As expected, BS176 was not
cytotoxic.
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Fig. 4.
S. flexneri is cytotoxic to macrophages
from both casp-3 and casp-11 knock-out
mice. A, peritoneal macrophages were isolated from
wild-type ( 
) or casp-3 / (
) mice or B,
from casp-11 / () mice. Macrophages were infected
either with M90T or BS176. Cytotoxicity was determined by measuring the
release of LDH after 3 h. Shigella is cytotoxic to macrophages
from both casp-3 / and casp-11 /
mice.
/ mice to the same extent. BS176 was not cytotoxic to
macrophages from either mice lineage. Since Casp-11 is induced after
LPS activation, we also tested LPS-activated macrophages from control
and casp 11 / mice. M90T induced cell death
with equal efficiency in both types of activated macrophages (data not shown).
/ as efficiently as wild type macrophages
(Fig. 5A), demonstrating that
Shigella-induced apoptosis is independent of p53. BS176 was unable to
kill either wild type or p53 /
macrophages.
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Fig. 5.
S. flexneri is cytotoxic to macrophages
from p53 knock-out mice and to RAW cells overexpressing
Bcl-2. A, peritoneal macrophages isolated from
wild-type ( ) or p53 / () mice and B,
RAW cells transfected with the vector control () or with the vector
encoding bcl-2 () were infected with either M90T or
BS176. C, RAW cells transfected with the vector control
() or with the vector encoding bcl-2 () were treated
with Etoposide for 24 h. Cytotoxicity was determined by measuring
the release of LDH. M90T is cytotoxic to macrophages from both
p53 knock-out mice and to RAW cells overexpressing Bcl-2.
However, Bcl-2 could protect RAW cells from Etoposide-induced
apoptosis.
DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
and IL-18 (15, 16). Interestingly, these cytokines do not encode a
signal sequence and it is still unknown how they reach the
extracellular space after cleavage by Casp-1 (40, 41). The link between
apoptosis and inflammation by Casp-1 is surprising, since apoptosis is
classically considered to be a immunologically silent cell death (42).
Indeed, during development and homeostasis, numerous apoptotic events
occur without eliciting inflammation. The role of macrophage cell death
in the release of IL-1 and IL-18 is still not understood.
/ mice develop normally and
macrophages from these mice undergo apoptosis induced by several
stimuli (18, 19). These results suggest either that Casp-1 function is
redundant or is not required in the pathways leading to apoptosis in
development or in response to certain stimuli.
/
mice. Casp-11 is closely related to casp-1 and is classified
within the same caspase family (Fig. 4). IpaB bound both the zymogen of
Casp-1 as well as a protein of 30 kDa that could be an intermediate
maturation product.
indicates that the mature Casp-1 is active during infection (Fig. 2C). The irreversible inhibitor YVAD-cmk has higher
affinity for Casp-1 than for other caspases. This inhibitor blocks
Shigella-induced apoptosis (9) and also Casp-1 activity as shown by its
inhibition of IL-1 cleavage (Fig. 2D). Surprisingly, YVAD
also inhibits Casp-1 maturation (Fig. 2B). Although in cell
lysates, YVAD has a significantly higher affinity for mature Casp-1
than for its zymogen (43), the affinity of the Casp-1 zymogen for YVAD
in vivo is not known. Our results could indicate that in
Shigella infections: 1) YVAD inhibits the autocatalytic activity of the zymogen, 2) that mature Casp-1 activity is necessary to activate the
Casp-1 precursor, or 3) that a different caspase with high affinity for
YVAD is necessary for Casp-1 activation.
/ are susceptible to
other apoptotic stimuli (18). Although Casp-3 is implicated in many different apoptotic processes and is thought to be a downstream effector protease in a caspase cascade (11), it is not necessary for
Shigella killing (Fig. 4).
/ mice are insensitive to Shigella cytotoxicity (Fig. 4). The independence of
Shigella-induced apoptosis of Casp-11 is not surprising since both
naive and activated wild type or casp-11 macrophages are susceptible to Shigella-induced cell death (Figs. 2 and 4, Ref. 33),
although Casp-11 is up-regulated only in activated macrophages. Thus,
Shigella induces a unique form of apoptosis which is dependent on
Casp-1, does not require Casp-3, and bypasses the requirement for
Casp-11 activation.
. Pretreatment of
macrophages with IL-1 receptor antagonist, a natural competitor of
IL-1, does not prevent Shigella-induced
apoptosis.2 Furthermore,
filtered, IL-1 rich, supernatants of macrophages infected with Shigella
are not cytotoxic to naive
macrophages.3 These results
strongly suggest that Casp-1-induced apoptosis does not function
through IL-1.
/ macrophages to Shigella is not surprising, since
S. flexneri-induced apoptosis does not require transcription
or translation (6) and can occur at any point during the cell
cycle.3 Bcl-2, and other members of the same family, are
thought to be pore forming proteins that regulate traffic across
mitochondrial and probably other membranes (38). Bcl-2 (Fig.
5B) and Bcl-xL (data not shown), two
anti-apoptotic members of the Bcl-2 family, do not inhibit
Shigella-induced cell death. These data suggest that Bcl-2 acts
upstream of Casp-1 activation or that these anti-apoptotic proteins
cannot inhibit Casp-1-dependent cell death in this system.
as well as
apoptosis effector proteins. Direct binding of IpaB to Casp-1 bypasses
the need for a signal transduction pathway and caspases upstream of
Casp-1, such as Casp-11. Although we have ruled out the possibility of
Casp-3 being a necessary substrate for Casp-1 in Shigella-induced
apoptosis, it is still unclear whether other downstream caspases are
needed. Whether this pathogenic mechanism is specific for Shigella
infections remains to be determined. Salmonella, however, encodes a
protein homologous to IpaB, the Salmonella invasion protein (Sip)B
which also binds to
Casp-1.4
does not encode a signal sequence, apoptosis might allow for
the release of the mature cytokine into the extracellular milieu. This
model of proinflammatory apoptosis is particularly attractive in
Shigellosis, in which the bacteria activate a
Casp-1-dependent apoptosis and IL-1 is the triggering
cytokine of the inflammatory response. Furthermore, using dominant
negative Casp-1 transgenic mice, it was recently shown that this enzyme
is required for ischemic brain injury (45), suggesting a role for
Casp-1 in other acute pathologies.
ACKNOWLEDGEMENTS
FOOTNOTES
Supported by a fellowship from the Swiss National Science Foundation.
REFERENCES
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Abstract
Introduction
Procedures
Results
Discussion
References
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
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