Anthrax lethal toxin and Salmonella elicit the
common cell death pathway of caspase-1-dependent
pyroptosis via distinct mechanisms
Susan L. Fink*, Tessa Bergsbaken†, and Brad T. Cookson†‡§
*Molecular and Cellular Biology Program, Departments of†Microbiology and‡Laboratory Medicine, University of Washington, Seattle, WA 98195
Edited by Charles A. Dinarello, University of Colorado Health Sciences Center, Denver, CO, and approved January 30, 2008 (received for review
August 5, 2007)
inflammatory cytokines. In Salmonella-infected macrophages,
caspase-1 also mediates a pathway of proinflammatory pro-
grammed cell death termed ‘‘pyroptosis.’’ We demonstrate active
caspase-1 diffusely distributed in the cytoplasm and localized in
discrete foci within macrophages responding to either Salmonella
infection or intoxication by Bacillus anthracis lethal toxin (LT). Both
stimuli triggered caspase-1-dependent lysis in macrophages and
dendritic cells. Activation of caspase-1 by LT required binding,
uptake, and endosome acidification to mediate translocation of
lethal factor (LF) into the host cell cytosol. Catalytically active LF
cleaved cytosolic substrates and activated caspase-1 by a mecha-
nism involving proteasome activity and potassium efflux. LT acti-
vation of caspase-1 is known to require the inflammasome adapter
Nalp1. In contrast, Salmonella infection activated caspase-1
through an independent pathway requiring the inflammasome
adapter Ipaf. These distinct mechanisms of caspase-1 activation
converged on a common pathway of caspase-1-dependent cell
death featuring DNA cleavage, cytokine activation, and, ulti-
mately, cell lysis resulting from the formation of membrane pores
between 1.1 and 2.4 nm in diameter and pathological ion fluxes
that can be blocked by glycine. These findings demonstrate that
distinct activation pathways elicit the conserved cell death effector
mechanism of caspase-1-mediated pyroptosis and support the
notion that this pathway of proinflammatory programmed cell
death is broadly relevant to cell death and inflammation invoked
by diverse stimuli.
single-cell analysis ? apoptosis ? inflammasome ? inflammation ?
programmed cell death
tory cytokines. Caspase-1 has emerged as a critical determinant
of both pathological inflammation and resistance to infectious
diseases. Notably, caspase-1-deficient mice are protected from
endotoxic shock (1, 2), ischemic injury (3, 4), and inflammatory
salmonellosis (6, 7). Salmonella enterica serovar Typhimurium is
a pathogen that invades host macrophages and stimulates
caspase-1-dependent cell death (8). Salmonella-infected macro-
phages produce activated IL-1? and IL-18 and undergo rapid
lysis with the release of inflammatory intracellular contents, and
thus the term ‘‘pyroptosis’’ is used to describe this form of
proinflammatory cell death (9).
Activation of caspase-1 occurs via induced proximity in in-
flammasomes or pyroptosomes, which are protein complexes
analogous to the apoptosis-inducing apoptosome (10, 11). In-
flammasomes contain NOD-like receptor (NLR) family pro-
teins, which are cytosolic pattern-recognition receptors stimu-
lated by infectious agents and endogenous danger signals.
Salmonella-induced activation of caspase-1 requires the host
NLR protein Ipaf, as well as the bacterial type III secretion
system (T3SS) and flagellin (10). The NLR protein Nalp3
he caspase-1 protease causes cell death and cleaves the
precursors of IL-1? and IL-18, producing mature inflamma-
activates caspase-1 in response to extracellular ATP binding to
cell surface P2X7receptors (12), and Nalp1 is required for the
activation of caspase-1 and macrophage death in response to
anthrax lethal toxin (LT), a critical virulence factor of Bacillus
anthracis (13). LT entry into host cells has been elegantly
characterized, yet the mechanism(s) of cytotoxicity are incom-
pletely defined. Further, it is unclear how inflammasomes are
regulated or how different stimuli activate caspase-1. Events
downstream of caspase-1 activation, other than IL-1? and IL-18
activation, have only recently been described for Salmonella
infection (14). Here we demonstrate that LT intoxication and
which converge to cause host cell death using an apparently
conserved program of caspase-1-mediated pyroptosis. Together
with the observations that caspase-1 is involved in a wide variety
of pathological conditions, our data support the idea that the
proinflammatory programmed cell death pathway of pyroptosis
is of broad biological relevance to cell death and inflammation
triggered by diverse stimuli.
LT and Salmonella Stimulate Caspase-1-Dependent Death of Macro-
phages and Dendritic Cells (DCs). We examined the activation of
caspase-1 by staining macrophages with a fluorescent peptide
(FAM-YVAD-FMK) that binds specifically and irreversibly to
active caspase-1. Whereas mock-infected macrophages have no
detectable active caspase-1 (Fig. 1A), those infected with Sal-
monella contain active caspase-1 in large bright foci and diffusely
throughout the cell [Fig. 1B and supporting information (SI) Fig.
9]. The localization of active caspase-1 in LT-treated macro-
phages was strikingly similar, with discrete brightly staining foci
and diffuse active caspase-1 (Fig. 1C). LT has been suggested to
stimulate macrophage apoptosis (15). However, a highly sensi-
tive assay did not detect activity of the central apoptotic effector,
caspase-3, in LT-treated macrophages (SI Fig. 10 and SI Mate-
rials and Methods).
LT and Salmonella are both cytotoxic for macrophages (8, 16),
and we confirmed cell lysis by measuring the release of cytosolic
lactate dehydrogenase (LDH). LT-intoxicated and Salmonella-
infected macrophages underwent caspase-1-dependent lysis that
was prevented by the specific caspase-1 inhibitor YVAD (17),
but not the negative control inhibitor zFA (Fig. 1D and SI Fig.
11). Caspase-1-independent release of LDH by cells treated with
H2O2was not affected by YVAD or zFA (Fig. 1D). DCs also are
Author contributions: S.L.F., T.B., and B.T.C. designed research; S.L.F. and T.B. performed
research; S.L.F., T.B., and B.T.C. analyzed data; and S.L.F. and B.T.C. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
§To whom correspondence should be addressed. E-mail: email@example.com.
This article contains supporting information online at www.pnas.org/cgi/content/full/
© 2008 by The National Academy of Sciences of the USA
March 18, 2008 ?
vol. 105 ?
no. 11 www.pnas.org?cgi?doi?10.1073?pnas.0707370105
targets of LT (18) and Salmonella (19), and lysis of LT-
intoxicated and Salmonella-infected DCs was blocked specifi-
cally by YVAD (Fig. 1E), indicating caspase-1-dependent death
in both cell types.
LT consists of two proteins, protective antigen (PA) and lethal
factor (LF). PA binds cell surface receptors, triggering receptor-
mediated endocytosis of the toxin complex (20). Endosome
acidification causes a conformational change in PA, allowing LF
translocation into the host cell cytosol, where it acts as a
metalloprotease cleaving several mitogen-activated protein ki-
nase kinases (MEKs), including MEK3 (21). Ammonium chlo-
ride (NH4Cl) prevents the acidification of intracellular compart-
ments, traps LF within membrane-bound endosomes (16), and
prevents caspase-1 activation in response to LT (Fig. 2A).
with LT, the production of mature IL-18 was inhibited by NH4Cl
(Fig. 2B). LT containing catalytically inactive E687C mutant LF
did not activate caspase-1 (Fig. 2 A and B). Therefore, although
extracellular ATP binding the cell surface P2X7receptor acti-
vates caspase-1 (12), PA interactions with cellular receptors
alone are not sufficient to trigger caspase-1 activation. LF
translocation from the endosome into the host cell cytosol and
LF catalytic activity are both required for LT-induced caspase-1
activation. Similarly, Salmonella entry into macrophages and
delivery of stimulatory ligand(s) to the cytosol via the bacterial
T3SS are required for cell death and caspase-1 activation (10).
In contrast, although acidification of the Salmonella-containing
phagosome is necessary for the expression of bacterial virulence
genes (22), Salmonella-infected macrophages activated
caspase-1 and secreted mature IL-18 in the presence of NH4Cl
(Fig. 2 A and B). Thus, we sought to investigate the mechanism
of LT-induced caspase-1 activation and cell death, with the aim
of testing the possibility that different stimulatory pathways,
used by Salmonella and LT, trigger caspase-1 activation and
evoke a conserved program causing cell death.
Caspase-1 Activation by LT Is Ca2?-Dependent. Ca2?is involved in
LT-induced cytotoxicity because both Ca2?-free culture medium
and Ca2?channel blockers protect macrophages from LT (23),
suggesting a role for Ca2?in caspase-1 activation. Ca2?-free
medium and the Ca2?channel blocker, verapamil, both pre-
vented caspase-1 activation after LT treatment, but not Salmo-
nella infection (Fig. 3A), indicating that Ca2?is not always
involved in caspase-1 activation, but is necessary for the pathway
induced by LT. To address the mechanism of this Ca2?require-
ment, we examined cleavage of LF substrate, MEK3. Catalyti-
cally active LF failed to cleave MEK3 in macrophages treated
with NH4Cl (Fig. 3B Upper). However, MEK3 cleavage was
restored in macrophages treated with LT and NH4Cl after
cellular disruption with detergent (Fig. 3B Lower), consistent
with NH4Cl allowing LT uptake, but trapping LF in a detergent-
soluble membrane-bound compartment (16). Similarly, vera-
pamil blocked MEK3 proteolysis in LT-treated macrophages
(Fig. 3B Upper) unless the cells were treated with detergent to
fore, like NH4Cl, verapamil rescues cells from LT-induced
caspase-1 activation by interfering with LF translocation to the
cytosol perhaps by disrupting the trafficking of intracellular
compartments (24). In contrast, Ca2?-free medium completely
prevented MEK3 proteolysis in LT-treated macrophages, even
in detergent lysates (Fig. 3B). LF added directly to these lysates
cleaved MEK3 (Fig. 3B), indicating that extracellular Ca2?is
necessary for the processes leading to significant LT uptake by
primary bone marrow-derived macrophages (BMDMs). Inter-
estingly, extracellular Ca2?is not required for LT uptake by the
J774A.1 macrophage-like cell line (23). Therefore, caspase-1
activation by LT requires extracellular Ca2?to permit LT uptake
by BMDM and translocation of catalytically active LF into the
cytosol, which is blocked by both NH4Cl and the Ca2?channel
blocker, verapamil. In contrast, Salmonella infection activates
caspase-1 independently of extracellular Ca2?and Ca2?chan-
nels sensitive to verapamil.
macrophages and DCs. (A–C) Macrophages were treated with PBS (A), S.
typhimurium (S.T) (B), or LT (C), and active caspase-1 was identified by FAM-
YVAD staining (green). Macrophages counterstained with TOPRO-3 (blue)
active caspase-1 are indicated by filled arrowheads. (B) Bacteria stained by
TOPRO-3 are indicated by open arrowheads. (D and E) Macrophages (M?) or
DCs were treated with LT or S.T in the presence of the specific caspase-1
inhibitor YVAD or the negative control inhibitor zFA. LDH released by dying
cells was quantified; means ? SD are shown.*, P ? 0.05 versus medium.
Lethal toxin and Salmonella stimulate caspase-1-dependent lysis of
activity to stimulate caspase-1. Macrophages were treated with PBS, LT, LT
containing catalytically inactive E687C mutant LF, or S. typhimurium (S.T) in
the presence or absence of 10 mM NH4Cl to inhibit the acidification of
intracellular compartments. (A) Macrophages containing active caspase-1
were identified by FAM-YVAD staining; means ? SD are shown.*, P ? 0.05
versus LT. (B) Mature IL-18 was detected in supernatants by Western blot.
LT requires acidification of intracellular compartments and catalytic
Fink et al. PNAS ?
March 18, 2008 ?
vol. 105 ?
no. 11 ?
Proteasome Activity and Potassium Efflux Are Required for Caspase-1
Activation by LT Subsequent to LF-Mediated Proteolytic Events. LT-
induced cytotoxicity requires proteasome-mediated protein deg-
radation (25). In addition, proteasome-dependent degradation
of the Raf-1 kinase occurs in Salmonella-infected macrophages
and has been suggested to be involved in the death of these cells
(26). The proteasome inhibitors MG-132 and lactacystin pre-
vented caspase-1 activation (Fig. 4A) and secretion of mature
IL-18 (Fig. 4B) in response to LT intoxication, but not Salmo-
nella infection. Neither inhibitor altered proteolysis of the LF
for MEK1 (25). These findings indicate that a proteasome-
dependent process mediates caspase-1 activation after LT up-
take and LF proteolysis of cytosolic proteins, whereas Salmo-
nella infection activates caspase-1 independently of proteasome
activity. A well described function of the proteasome is NF-?B
activation, and NF-?B potentiates caspase-1 activation by ATP-
mediated P2X7 receptor stimulation (27). The inhibitor Bay
11-7085, which blocks NF-?B activation (27), did not prevent
lysis of LT-treated cells (data not shown), indicating that LT-
stimulated caspase-1 activation and cell lysis require other
P2X7 receptor ligation stimulates caspase-1 by causing the
efflux of intracellular potassium, and caspase-1 activation by this
stimulus is prevented in cell culture medium with sodium
replaced by potassium to eliminate the gradient for potassium
efflux (28, 29). To test the hypothesis that potassium efflux also
is required for caspase-1 activation induced by LT intoxication
and Salmonella infection, macrophages were treated in medium
with sodium replaced by potassium. Elimination of the gradient
for potassium efflux selectively prevented caspase-1 activation
(Fig. 5A) and secretion of mature IL-18 (Fig. 5B) in response to
LT intoxication, but not Salmonella infection. This finding
indicates that potassium efflux is not universally required for
inflammasome formation or caspase-1 activation, but is differ-
entially involved in caspase-1 stimulation by LT intoxication.
Macrophages treated with LT in medium with sodium replaced
by potassium contained cleaved MEK3 (Fig. 5C), indicating that
the uptake and proteolytic function of LF were not affected.
Rather, potassium efflux is required for caspase-1 activation
downstream of LF substrate proteolysis.
Pyroptosis Stimulated by LT and Salmonella Features a Common
Mechanism of Lysis Mediated by Pore Formation. We previously
described caspase-1-dependent pores in Salmonella-infected
macrophages that contribute to the lysis of these cells (14).
Plasma membrane pores dissipate cellular ionic gradients, but
retain large cytoplasmic constituents, leading to net increased
osmotic pressure, water influx, cell swelling, and osmotic lysis
(30). Osmotic lysis is prevented by osmoprotectants with mo-
lecular diameters greater than the functional diameter of the
pores (31), and, importantly, osmoprotection is not observed
with molecular diameters of ?2.4 nm (33) rescued both LT-
intoxicated and Salmonella-infected macrophages from lysis,
whereas molecules ?1.1 nm had no effect; none prevented lysis
of H2O2-treated controls (Fig. 6A). Osmoprotective PEG 1450
and PEG 2000 did not inhibit caspase-1 activation, demonstrat-
ing that their effect is specific for lysis (Fig. 6B). Thus, caspase-
1-dependent lysis stimulated by both LT intoxication and Sal-
action and LF translocation into the cytosol. Macrophages were treated with
containing 150 ?M verapamil, a Ca2?channel blocker. (A) Macrophages
containing active caspase-1 were identified by FAM-YVAD staining; means ?
SD are shown.*, P ? 0.05 versus medium. (B) Cleaved LF substrate MEK3
(arrow) in LT-intoxicated macrophages was detected by Western blot. Where
LF and cleaved MEK3 was detected by Western blot in the lysates.
Calcium is required for caspase-1 activation by LT: target cell inter-
LF-mediated proteolytic events. (A) Macrophages treated with LT or S. typhi-
murium (S.T) in the presence of proteasome inhibitors (1 ?M MG-132 or 5 ?M
lactacystin) were examined for active caspase-1 by FAM-YVAD staining;
means ? SD are shown.*, P ? 0.05 versus medium. (B) Mature IL-18 was
detected in supernatants by Western blot. (C) Cleaved MEK3 (arrow) in LT
intoxicated macrophages was detected as in Fig. 3.
with LT or S. typhimurium (S.T) in standard medium containing high Na?or
modified medium containing Na?replaced by K?. (A) Macrophages contain-
ing active caspase-1 were identified by FAM-YVAD staining; means ? SD are
shown.*, P ? 0.05 versus Na?medium. (B) Mature IL-18 was detected in
supernatants by Western blot. (C) Cleaved MEK3 (arrow) in LT-intoxicated
macrophages was detected as in Fig. 3.
Potassium efflux is required for caspase-1 activation by LT, but is
www.pnas.org?cgi?doi?10.1073?pnas.0707370105Fink et al.
the formation of plasma membrane pores between 1.1 and 2.4
nm in diameter.
Osmotic lysis due to membrane pores results from cellular loss
of ionic equilibrium (31), where pathological ion fluxes can be
nonspecifically inhibited by the cytoprotective agent glycine (34,
35). Glycine inhibits swelling and lysis of Salmonella-infected
macrophages, without preventing formation of plasma mem-
brane pores (14), and glycine also prevented lysis of LT-treated
macrophages (Fig. 6C). Caspase-1 activation in response to both
LT and Salmonella was unaffected by glycine (Fig. 6B). Together
these data indicate that a common pathway of caspase-1-
dependent lysis during pyroptosis is mediated by membrane
pores between 1.1 and 2.4 nm in diameter and pathological ion
fluxes that can be blocked by glycine.
Caspase-1-Dependent DNA Cleavage Is a Common Feature of Pyrop-
tosis Stimulated by LT and Salmonella. Degradation of chromo-
somal DNA is a well recognized feature of apoptosis, but also
occurs during pyroptosis of Salmonella-infected macrophages
(35). Using the TUNEL reaction, we found damaged DNA in
LT-treated macrophages (Fig. 7), which was completely pre-
vented by YVAD. Thus, caspase-1-dependent DNA cleavage is
an additional common consequence of pyroptosis in response to
both LT intoxication and Salmonella infection.
Our findings describe distinct mechanisms by which anthrax LT
intoxication and Salmonella infection activate host cell caspase-1
to cause cell death by converging on the common pathway of
caspase-1-mediated pyroptosis. Although exogenous ATP and
anthrax LT bind specific macrophage surface receptors and
activate caspase-1, activation by LT requires delivery of its
metalloproteinase LF subunit into the cytoplasm. Ca2?-
dependent uptake of the LT complex precedes LF cytoplasmic
translocation, which requires endosome acidification and is
inhibited by the Ca2?channel blocker, verapamil. LF proteolytic
activity is necessary, but not sufficient. In addition to cleaving
requires proteasome activity and potassium efflux (Fig. 8) and
the inflammasome protein Nalp1 (13). Salmonella activates
caspase-1 by an independent pathway (Fig. 8) that requires the
inflammasome protein Ipaf (10). Caspase-1 activated in re-
common pathway of caspase-1-dependent pyroptosis. (Upper) The LT com-
plex consisting of PA and LF is taken up by macrophages in a Ca2?-dependent
manner. Endosome acidification, which is blocked by NH4Cl, triggers a con-
formational change in PA, allowing translocation of LF into the cytosol. The
proteolytically cleaves MEK and other substrates, after which caspase-1 acti-
protein Nalp1. Salmonella infection stimulates caspase-1 by an independent
(T3SS) and the inflammasome protein Ipaf. (Lower) Caspase-1 activated by
both stimuli mediates a common pathway of pyroptosis: cell death featuring
DNA fragmentation, secretion of activated inflammatory cytokines, and lytic
release of inflammatory intracellular contents mediated by the formation of
membrane pores between 1.1 and 2.4 nm in diameter. Osmotic lysis during
pyroptosis is blocked by osmoprotectants and the cytoprotective agent
Lethal toxin and Salmonella use distinct mechanisms to elicit the
mediated by pore formation. (A–C) Macrophages were treated with LT, S.
typhimurium (S.T), or 5 mM H2O2 in the presence of osmoprotectants of
varying sizes (A and B) or glycine (B and C), which nonspecifically inhibits ion
fluxes. (A and C) LDH released by dying cells was quantified; means ? SD are
shown.*, P ? 0.05 versus medium. (B) Macrophages containing active
caspase-1 were identified by FAM-YVAD staining; means ? SD are shown.
Lethal toxin and Salmonella stimulate a common mechanism of lysis
rophages and during Salmonella infection. Macrophages were treated with
PBS, LT, or S. typhimurium (S.T) in the presence of the specific caspase-1
inhibitor YVAD or the negative control inhibitor zFA. Macrophages contain-
*, P ? 0.05 versus medium.
Caspase-1-dependent DNA fragmentation occurs in LT-treated mac-
Fink et al.PNAS ?
March 18, 2008 ?
vol. 105 ?
no. 11 ?
sponse to LT intoxication and Salmonella infection exhibits the
same pattern of localization diffusely throughout the cell and in
discrete, brightly staining foci. These foci may represent active
caspase-1 localized within Ipaf- and Nalp1-containing inflam-
masomes, respectively. Similar structures containing oligomers
of the adaptor protein ASC have recently been visualized and
termed ‘‘pyroptosomes’’ (11). Caspase-1 activated by both stim-
uli mediates a common pathway of caspase-1-dependent cell
death, or pyroptosis, featuring DNA cleavage, cytokine activa-
tion, and lysis mediated by the formation of membrane pores
between 1.1 and 2.4 nm in diameter. Osmotic lysis during
pyroptosis is blocked by osmoprotectants and glycine. This
process is not limited to macrophages; both LT intoxication and
Although Ca2?and potassium fluxes are necessary for
caspase-1 activation in response to some stimuli (11, 36, 37), the
ability of Salmonella infection to activate caspase-1 indepen-
dently of these ion fluxes demonstrates that they are not
absolutely required for inflammasome formation as previously
hypothesized. However, LT activation of caspase-1 through
Nalp1 requires potassium efflux (Fig. 5), and the Nalp3 inflam-
masome (12) and the ASC pyroptosome (11) also respond to
potassium efflux-inducing agents, suggesting that potassium
efflux may be a common signal for both Nalp-containing in-
flammasomes and the pyroptosome. The route of potassium
efflux during LT intoxication is unclear. Although potassium
efflux can occur through the P2X7receptor ion channel, a role
for this channel in LT intoxication was previously excluded (38).
Our results suggest that it is unlikely that Nalp1 is directly
activated by recognition of LF as a ligand because catalytically
inactive E687C mutant LF shares a virtually identical structure
caspase-1. Additionally, the inhibition of potassium efflux or
proteasome activity prevents caspase-1 activation, but not MEK
proteolysis, demonstrating that the presence of cytosolic LF and
MEK cleavage are not sufficient to stimulate the inflammasome.
LF may cleave additional, as yet unknown, targets, leading to the
degradation of Nalp1 inhibitors or the production of activating
factors that trigger inflammasome function.
Apoptosis was initially described simply based on common
morphological features of dying cells observed in different
physiological contexts (40). It is now well recognized that
apoptosis represents a conserved pathway of cell death stimu-
lated by diverse molecular mechanisms. The extrinsic, intrinsic,
and ER stress pathways each includes a plethora of specific
signaling mechanisms that all converge on the common outcome
of apoptosis (41). Our findings demonstrate that pyroptosis also
is a conserved program of cell death occurring in response to
distinct signals. In contrast to the noninflammatory outcome of
apoptosis, pyroptosis is characterized by activation of the in-
flammatory cytokines, IL-1? and IL-18, and lysis with release of
inflammatory intracellular contents.
In vivo, caspase-1 activation in response to innate immune
recognition of microbe-associated patterns helps provide host
resistance to infectious pathogens, including Salmonella (6, 7),
Shigella (42), Legionella (43, 44), Francisella (45), Listeria (46),
and Yersinia (47). Caspase-1 also provides host-protective func-
tions during B. anthracis infection; human DCs and alvelovar
macrophages produce IL-1? in response to spore infection (48,
49). IL-1? production in BALB/c mice correlates with early
control of bacterial dissemination (50). Correspondingly, LT-
deficient mutants exhibit greater initial dissemination after
murine inoculation with spores or vegetative bacilli (51). In
addition to its protective role in innate immunity, caspase-1 plays
a role in the pathogenesis of conditions featuring inflammation
and cell death, including neurodegenerative diseases (52), in-
flammatory bowel disease (5), endotoxic shock (1, 2), myocar-
dial infarction (3, 53), cerebral ischemia (54), and renal injury (4,
55). Therefore, the conserved proinflammatory programmed
cell death pathway of pyroptosis is likely to have broad biological
Materials and Methods
Cell Culture. From BALB/c femur exudates, BMDMs were cultured for 7 days in
verapamil, 1 ?M MG-132, 5 ?M lactacystin, 5 mM glycine, or 60 mM osmopro-
KHCO3, and 0.906 mM KH2PO4; control medium contained 110.34 mM NaCl, 5.8
mM KCl, 44.1 mM NaHCO3, and 0.906 mM NaH2PO4; both contained 1.8 mM
mM Hepes, 0.2 mg?ml?1L-glutamine, 0.05 mM ?-mercaptoethanol, 25? MEM
PA and 1 ?g?ml?1LF or catalytically inactive E687C mutant LF (List Biological) for
LDH ? spontaneous LDH).
Fluorescence Microscopy. Macrophages stained for active caspase-1 (FAM-
YVAD-FMK; Immunochemistry Technologies) on glass coverslips were fixed
and counterstained with TO-PRO-3. TUNEL staining of damaged DNA was
performed as described previously (35), and cells were counterstained with
TO-PRO-3. At least 295 cells were examined for each experimental condition
cell population. Images were reduced in size with Adobe Photoshop.
Western Analysis. To analyze MEK3 cleavage in intact cells treated with LT for
For detergent-treated cells, where MEK3 cleavage is examined in cellular
lysates after releasing any active LF trapped in membranous compartments,
macrophages treated with LT for 1 h were incubated on ice for 5 min in 1%
Triton X-100; adding SDS sample buffer stopped proteolytic activity. For IL-18
immunoblotting, serum-free macrophage supernatant was concentrated by
diafiltration. Proteins were separated by SDS/PAGE, transferred to nitrocellu-
lose membranes, and interrogated with anti-MEK-3 antibody or anti-IL-18
antibody (Santa Cruz Biotechnology).
Statistics. Data were analyzed by unpaired two-tailed Student’s t test.
ACKNOWLEDGMENTS. We thank Matthew Johnson for technical assistance;
was supported by National Institutes of Health Grants AI47242 and P50
HG02360, Poncin and Achievement Rewards for College Scientist Fellowships
(to S.L.F.), and National Institute of General Medical Sciences Public Health
Service National Research Service Award Grant T32 GM07270 (to T.B.).
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