Potentiation of Caspase-1 Activation by the P2X7 Receptor Is
Dependent on TLR Signals and Requires NF-?B-Driven
J. Michelle Kahlenberg,* Kathleen C. Lundberg,†Sylvia B. Kertesy,†Yan Qu,‡and
George R. Dubyak2†
The proinflammatory cytokines IL-1? and IL-18 are inactive until cleaved by the enzyme caspase-1. Stimulation of the P2X7
receptor (P2X7R), an ATP-gated ion channel, triggers rapid activation of caspase-1. In this study we demonstrate that pretreat-
ment of primary and Bac1 murine macrophages with TLR agonists is required for caspase-1 activation by P2X7R but it is not
required for activation of the receptor itself. Caspase-1 activation by nigericin, a K?/H?ionophore, similarly requires LPS
priming. This priming by LPS is dependent on protein synthesis, given that cyclohexamide blocks the ability of LPS to prime
macrophages for activation of caspase-1 by the P2X7R. This protein synthesis is likely mediated by NF-?B, as pretreatment of cells
with the proteasome inhibitor MG132, or the I?B kinase inhibitor Bay 11-7085 before LPS stimulation blocks the ability of LPS
to potentiate the activation of caspase-1 by the P2X7R. Thus, caspase-1 regulation in macrophages requires inflammatory stimuli
that signal through the TLRs to up-regulate gene products required for activation of the caspase-1 processing machinery in
response to K?-releasing stimuli such as ATP. The Journal of Immunology, 2005, 175: 7611–7622.
To interact with its receptor, IL-1? must first be cleaved from its
33-kDa pro-form into the 17-kDa active mature cytokine mIL-1?.
Caspase-1 is required for this cleavage because caspase-1 knock-
out macrophages are unable to induce IL-1? cleavage in response
to any activating stimuli (2, 3). Given the essential role of
caspase-1 in this process, the mechanisms underlying caspase-1
activation have been intensively studied but remain only partially
defined. Caspase-1 is constitutively expressed as procaspase-1, a
low-activity zymogen that contains an N-terminal caspase recruit-
ment domain (CARD)3essential for caspase-1 activation in vivo.
Scaffolding proteins with similar CARD, including the adaptor
molecule ASC (apoptosis-associated speck-like protein containing
a CARD), bind to and oligomerize procaspase-1 within protein
complexes termed the inflammasomes, which facilitate caspase-1
autocleavage into p20 and p10 fragments from the C terminus
(4–12). These free p20 and p10 fragments assemble into an active
heterotetramer that acts as a highly efficient IL-1? converting/
cleaving enzyme or ICE (12, 13).
aspase-1 is the activating enzyme for the proinflamma-
tory cytokines IL-1? and IL-18, which play an important
role in inflammatory disease, fever, and septic shock (1).
Although constitutively expressed, procaspase-1 remains inac-
tive in the cytoplasm until inflammatory effector cells, such as
monocytes and macrophages, receive appropriate stimuli. In these
cells, K?release stimuli induce rapid and robust activation of
caspase-1, resulting in the processing and release of mIL-1? (13–
18). One well-characterized physiological K?release stimulus is
extracellular ATP activation of the P2X7 receptor (P2X7R). Short-
term (5 min) stimulation of macrophages with ATP results in the
processing and release of IL-1? to the extracellular medium within
15 min (13, 19, 20). However, because IL-1? production is regu-
lated by induced expression as well as proteolysis, such studies
necessarily use LPS-primed monocyte/macrophages to up-regulate
the transcription and translation of IL-1? before acute stimulation
of P2X7R by ATP addition. Recent studies have demonstrated that
priming by LPS is also required for efficient caspase-1 activation
by ATP-gated P2X7R (4, 5). Thus, even though procaspase-1 and
P2X7R are constitutively expressed in non-LPS primed cells, LPS
priming is required to facilitate coupling between these two signal
transduction proteins. Long-term stimulation of monocytes with
LPS in the absence of ATP also induces a slowly developing ac-
tivation of caspase-1 (16, 17, 21–23); recent data indicate that this
action may reflect muramyl dipeptide (MDP) accumulation sec-
ondary to intracellular metabolism of the peptidoglycan contami-
nants in most commercial LPS preparations (24). Regardless of the
mechanism, this slow activation by LPS and/or MDP over a time
course of several hours contrasts with the rapid (within 5 min)
activation of caspase-1 observed with P2X7R stimulation.
LPS signals by binding to a complex of proteins, including se-
rum LPS binding protein, MD-2, CD14, and the extracellular do-
main of TLR4. TLR4 activation induces recruitment of the adaptor
protein MyD88 to the receptor, which results in the formation of a
signaling complex that allows for activation of several kinase path-
ways as well as the nuclear translocation of the transcription factor
NF-?B secondary to ubiquitination and degradation of its cyto-
plasmic inhibitor I?B (25). One recent study has suggested that the
adaptor molecules MyD88 and Toll/IL-1R domain-containing
*Department of Pathology,†Department of Physiology and Biophysics, and‡Depart-
ment of Pharmacology, Case School of Medicine, Case Western Reserve University,
Cleveland, OH 44106
Received for publication June 7, 2005. Accepted for publication September 19, 2005.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1This work was supported by National Institutes of Health (NIH) Grant GM36387 (to
G.R.D.). This work was also supported by NIH Grant T32GM07250 to the Medical
Scientist Training Program of Case School of Medicine (to J.M.K.).
2Address correspondence and reprint requests to Dr. George R. Dubyak, Department
of Physiology and Biophysics, Case School of Medicine, Case Western Reserve Uni-
versity, Cleveland, OH 44106. E-mail address: email@example.com
3Abbreviations used in this paper: CARD, caspase recruitment domain; MDP, mu-
ramyl dipeptide; mIL-1?, mature IL-1?; BSS, basal salt solution; IKK, I?B kinase;
BMDM, bone marrow-derived macrophage; PAMP, pathogen-associated molecular
The Journal of Immunology
Copyright © 2005 by The American Association of Immunologists, Inc. 0022-1767/05/$02.00
adaptor inducing IFN-? are not required for LPS-mediated prim-
ing of ATP-mediated caspase-1 activation (4). However, it is un-
known what signaling mechanisms downstream of the TLR facil-
itate the rapid coupling of ATP-occupied P2X7R to the caspase-1
Given the complex interplay between LPS priming and P2X7R
activation of caspase-1, we have characterized how signal trans-
duction processes induced by short-term LPS priming facilitate the
activation of caspase-1 by the P2X7R in both primary mouse bone
marrow-derived macrophages (BMDM) and a murine macrophage
cell line model. We demonstrate that P2X7R activation of
caspase-1 is dependent on prestimulation of macrophages with
LPS or other TLR ligands. Moreover, this priming effect is medi-
ated by rapid LPS-induced protein synthesis, likely mediated by
the transcription factor NF-?B.
Materials and Methods
Reagents and Abs
Reagents from the following sources were used: lactacystin (Biomol),
MG132 (Biomol), Bay 11-7085 (Biomol), Escherichia coli LPS serotype
O1101:B4 (List Biological Laboratories), monophosphoryl E. coli F583
lipid A (Sigma-Aldrich), SB203580 (Biomol), SP600125 (Calbiochem),
(Bachem). Human and murine IL-1? ELISA Abs (M-421B-E, M-420B-B,
PM-425B, and MM-425B-B) were from Pierce. Anti-P2X7R was from
Alamone Laboratories. Anti-IL-1? used for Western blots (3ZD) was pro-
vided by the Biological Resources Branch of the National Cancer Institute,
Frederick Cancer Research and Development Center (Frederick, MD). An-
ti-phospho-c-Jun, anti-phospho-Akt, anti-Akt, anti-phospho-ERK1/2, and
anti-ERK1/2 Abs were from Cell Signaling. Other Abs were obtained from
Santa Cruz Biotechnology anti-mouse caspase-1 p10 rabbit polyclonal, anti-
I?B?, anti-actin, and all HRP-conjugated secondaries. The 19-kDa Mycobac-
terium tuberculosis lipoprotein was a gift of Dr. C. V. Harding, III (Case
Western Reserve University, Cleveland, OH). CpG DNA 1886 was a gift from
Dr. F. Heinzel (Case Western Reserve University, Cleveland, OH).
Bac1.2F5 (Bac1) murine macrophages were cultured as previously de-
scribed (26) in DMEM supplemented with 25% L cell-cultured medium,
15% calf serum, and 1% penicillin-streptomycin in the presence of 10%
CO2. Cells were split 1:3 onto culture dishes 2–3 days before experiments.
Mouse BMDM were obtained as previously described (27). Briefly, femurs
from BALB/c mice (6–10 wk) were isolated and the marrow was flushed
with 10 ml of DMEM. The cells were washed once with DMEM and then
plated and cultured for 9 days in DMEM (Sigma-Aldrich) supplemented
with 25% L cell-cultured medium, 15% calf serum (HyClone Laborato-
ries), and 1% penicillin-streptomycin (100 U/ml penicillin and 100 ?g/ml
streptomycin; Invitrogen Life Technologies) in the presence of 10% CO2.
The resulting macrophages were detached with PBS supplemented with 5
mM EDTA and 4 mg/ml lidocaine and replated in six-well or 24-well
dishes for subsequent experiments. All experiments involving the use of
mice were approved by the Institutional Animal Care and Use Committee
of Case Western Reserve University. COS-1 cells were cultured in DMEM
(Sigma-Aldrich) supplemented with 10% calf serum (HyClone Laborato-
ries) and 1% penicillin-streptomycin (100 U/ml penicillin and 100 ?g/ml
streptomycin; Invitrogen Life Technologies) in the presence of 10% CO2.
In vitro assay for processing of caspase-1 and IL-1?
This assay was performed as previously described (20). Briefly, 1 ? 108
macrophages were treated with 500 ng/ml LPS for 4 h. Following this, the
cells were bathed in a basal salt solution (BSS) containing 130 mM NaCl,
5 mM KCl, 20 mM HEPES, 5 mM glucose, 0.01% BSA, 1.5 mM CaCl2,
and 1.0 mM MgCl2for 10 min followed by 1 mM ATP treatment for 5 min
where indicated. The cells were washed once in PBS and resuspended in 1
ml buffer W (20 mM HEPES (pH 7.5), 10 mM KCl, 1.5 mM MgCl2, 1.0
mM EGTA, 1.0 mM EDTA) supplemented with 2 mM DTT, 2 ?g/ml
leupeptin, 100 ?g/ml PMSF, and 2.5 ?g/ml aprotinin. The cells were then
pelleted and all but ?50 ?l of the buffer was removed. The cells were then
allowed to swell for 10 min on ice and were subsequently lysed by 15
passages through a 22-gauge needle. Lysates were then centrifuged at
15,000 ? g for 15 min and the supernatant was removed into a new tube
and kept on ice. Protein concentrations were determined using the Bradford
assay (Bio-Rad) and protein levels were adjusted to 22 mg/ml using buffer
W. A total of 10 ?l of lysates were aliquoted into 1.5-ml tubes and were
placed at 30°C for the indicated times. Processing reactions were stopped
by adding an equal volume of 4? SDS-PAGE buffer. Lysates were run on
15% polyacrylamide gels and transferred to polyvinylidene difluoride (Mil-
lipore). Western blots were done with the following Ab concentrations:
IL-1? 5 ?g/ml, caspase-1 5 ?g/ml. To determine the effects of inhibitors
on LPS potentiation of P2X7R activation of caspase-1, primary or Bac1
macrophages were preincubated with the inhibitors for 30 min followed by
(without removal of the inhibitor) a 3.5–4 h stimulation with 0.5–1 ?g/ml
LPS. Cells were then washed and treated as described.
Bac1 macrophages were seeded 1 ? 106cells per well of a 12-well dish for
12–18 h before experiments. Where indicated, cells were preincubated with
various inhibitors for 30 min followed by treatment with 1 ?g/ml LPS for
a total incubation time of 4 h, following removal of the LPS priming
medium. The cells were washed once with PBS, bathed with 1 ml of BSS,
and then stimulated with 1 mM ATP for 2–30 min. The medium was
removed and the cells were lysed in 1 ml of 10% nitric acid. The intra-
cellular K content was quantified using atomic absorbance spectroscopy
and compared with standards. In experiments in which no inhibitor was
used, the cells were treated with or without 1 ?g/ml LPS for 4 h and
processed as described.
In vitro JNK assay
This reaction was conducted as previously described (26). Briefly, Bac1
macrophages were seeded at 2 ? 106per well. The cells were then treated
as previously indicated and the cells were lysed in a 0.1% Triton X-100
buffer (25 mM HEPES (pH 7.5), 300 mM NaCl, 1.5 mM MgCl2, 200 ?M
EDTA, 0.1% Triton X-100, 500 ?M DTT, 20 mM ?-glycerophosphate, 2
?g/ml leupeptin, 2.5 ?g/ml aprotinin). Lysates were then precipitated using
3 ?g of GST-jun agarose beads for 4 h at 4°C. Beads were then washed
twice in lysis buffer and once in 50 mM HEPES/1 mM DTT. Beads were
then incubated with 20 ?l of kinase buffer (25 mM Tris (pH 7.4), 0.5 mM
DTT, 10 mM MgCl2, 7.5 ?M ATP) for 30 min at room temperature. The
reactions were stopped with 20 ?l of 2? SDS-PAGE sample buffer. The
phosphorylation of c-jun was monitored by Western blot using an Ab spe-
cific for the phosphorylated form of c-jun.
Activation of I?B kinase (IKK)
Activation of IKK by LPS was assayed by measuring the rapid degradation
of I?B-?. Macrophages on six-well plates were preincubated with various
concentrations of MG132 or Bay 11-7045 for 30 min before stimulation
with LPS for 5–60 min. The cells were then lysed and processed for SDS-
PAGE and Western blotting as described for the caspase-1 activation ex-
periments. The transferred lysates were serially probed with anti-I?B-? and
Induction of caspase-1 activation and release
To determine the activation of caspase-1 within intact cells, Bac1 cells or
primary BMDM were stimulated with 0.5 or 1 ?g/ml LPS for up to 4 h.
Cells were then washed 1? with PBS and bathed in a sodium gluconate
balanced salt solution (130 mM sodium gluconate, 5 mM KCl, 20 mM
HEPES, 5 mM glucose, 0.01% BSA, 1.5 mM CaCl2, and 1.0 mM MgCl2)
for 10 min at 37°C. This process was followed by stimulation with 1 mM
ATP for 30 min. The extracellular medium was collected and the protein
was precipitated using TCA and redissolved in SDS-PAGE buffer. The
cells were lysed in SDS-PAGE buffer. Both samples were then resolved on
a 15% acrylamide SDS-PAGE gel and processed for Western blot analysis
of IL-1? and caspase-1 levels.
To determine the amount of IL-1? released after ATP stimulation, we used
a sandwich ELISA protocol as previously described (17). Briefly, 1 ? 106
Bac1 macrophages were seeded into a six-well plate. After overnight in-
cubation, the cells were primed with 500 ng/ml LPS or 500 ng/ml lipid A
for 4 h, washed once with PBS, and the medium was replaced with 1.0 ml
of BSS. Cells were then stimulated with 1 mM ATP for 30 min. The
medium was then removed and 1–50 ?l was added to a BSA-blocked
ELISA plate that had been coated overnight with 1 ?g/ml anti-murine
IL-1?. Biotin-conjugated IL-1? Ab was then added and the plates were
incubated at room temperature for 2 h. The plate was then washed and
incubated with HRP-conjugated streptavidin (Pierce) for 30 min and de-
veloped using tetramethyl benzidine as substrate. The absorbance measure-
ments were read with a Molecular Devices SoftMax Pro plate reader and
were compared with IL-1? standards.
7612P2X7R ACTIVATION OF CASPASE-1 REQUIRES ACTIVATION OF NF-?B
Cleavage of recombinant pro-IL-1? as a measure of caspase-1
activity in cell-free lysates
Confluent 10-cm plates of COS-1 cells were split 1:3 the night before
transfection. Transfection was done using 2 ?g of human pro-IL-1? DNA
(a gift from Dr. S. Mizel, Wake Forest University Baptist Medical Center,
Winston-Salem, NC) per plate and Effectene reagent from Qiagen accord-
ing to manufacturer’s protocol. COS-1 cells were then lysed in hypotonic
buffer W by shear force with a 22-gauge needle and the protein concen-
tration was adjusted to 15 mg/ml. Bac1 macrophages were treated and
prepared as described for the in vitro processing assay, but 18.5 ?g of
COS-1 lysate protein containing the overexpressed IL-1? was added to 990
?g (45 ?l per tube) of Bac1 lysate protein and then the mixed lysate was
aliquoted (6 ?l per tube) and placed at 30°C for the indicated times. As a
control, untransfected COS-1 lysate was also added to Bac1 lysates. To
stop processing, the reaction was diluted 1/5 with buffer W and placed on
ice. An ELISA specific for the human mIL-1? 17-kDa fragment was used
to measure the processed IL-1? as previously described.
Priming by TLR4 is required for caspase-1 activation by the
When intact monocytes or macrophages are stimulated by ATP to
process and assemble caspase-1 p10/p20 heterotetramers, these ac-
tive caspase-1 complexes are rapidly released to the extracellular
environment, making it difficult to detect active caspase-1 in cell
lysates (28). Fig. 1A illustrates this rapid P2X7R-dependent release
of active caspase-1 in the Bac1 murine macrophage model cell
line. When Bac1 macrophages primed with LPS for 4 h were stim-
ulated with 1 mM ATP for up to 30 min, only minor intracellular
accumulation of the caspase-1 p10 subunit was observed. This
observation contrasted with the marked accumulation of caspase-1
p10 in the extracellular medium after a 5 min lag period following
ATP addition. Because recent reports have suggested that LPS
and other TLR ligands are required for efficient activation of
caspase-1 by ATP stimulation of the P2X7R in peritoneal mac-
rophages (4, 5), we also tested the ability of ATP to induce
rapid release of active caspase-1 from parallel samples of Bac1
cells that were not LPS primed. In the absence of LPS priming,
minimal active caspase-1 p10 fragment was released into the
extracellular medium even after 30 min of ATP stimulation.
Significantly, LPS priming did not alter the intracellular level of
procaspase-1 (Fig. 1A) but did induce significant accumulation
of the pro-IL-1? substrate of caspase-1. Fig. 1B illustrates that
LPS priming was similarly required to facilitate the coupling of
P2X7R to caspase-1 activation and release in primary BMDM
from BALB/c mice. Moreover, near-maximal activation of
caspase-1 by ATP-occupied P2X7R was observed in macro-
phages primed with LPS for only 60 min; this rapid induction of
the coupling machinery preceded the significant accumulation
of intracellular pro-IL-1?.
The activation of P2X7R induces rapid K?efflux from mono-
cyte/macrophages and previous studies have demonstrated that this
K?loss is required for caspase-1 activation by ATP-occupied
P2X7 (13, 15, 20). Fig. 1C, left, demonstrates that ATP stimulation
induced equally effective release of intracellular K?from macro-
phages with or without LPS priming, indicating that LPS priming
does not modulate P2X7R activation by ATP per se. Western blot
experiments verified similar levels of P2X7R protein in control or
LPS-primed macrophages (see Figs. 4C and 5D). LPS priming also
did not affect the kinetics of this ATP-induced K?release (Fig. 1C,
Other pathogen-associated molecular patterns (PAMPs) that can
contaminate LPS, such as peptidoglycan, or metabolites of such
PAMPs, such as MDP, have recently been implicated in the slow
activation of caspase-1 induced by long-term stimulation with LPS
(24). Lipid A is free of bacterial polysaccharides and is the active
moiety of LPS recognized by TLR4. Significantly, priming of
Bac1 macrophages with either lipid A or LPS produced equivalent
potentiation of ATP-induced caspase-1 activation as indicated by
the cleavage and secretion of mIL-1? detected by either ELISA
(Fig. 1D) or Western blot (Fig. 1D, inset). This suggests that the
priming of P2X7R-induced caspase-1 activation can be elicited by
multiple TLR4 ligands and is unlikely to involve polysaccharide
contaminants of LPS.
LPS priming is a general requirement for caspase-1 activation
by K?release stimuli
As indicated in these experiments (Fig. 1), the analysis of
caspase-1 activation in intact macrophages is complicated by the
near-simultaneous export of the processed caspase-1 to the extra-
cellular compartment. It is unclear from such intact cell studies
whether LPS priming is required for the intracellular assembly of
the caspase-1 activation complexes in response to K?release stim-
uli or for the secretion of these assembled complexes into the ex-
tracellular medium. We recently described a novel method for de-
termining the activation state of caspase-1 using an in vitro
processing assay (20) in which intact cells are pretreated with an
activating stimulus (i.e., ATP) followed by immediate lysis in a
hypotonic buffer and subsequent incubation of the cell-free lysates
at 30°C for various times. The kinetics of IL-1? or caspase-1 pro-
cessing in vitro reflects the activation state of caspase-1 within
assembled inflammasome complexes at the time of cell lysis. This
permits dissociation of caspase-1 activation signals from the se-
cretion of active caspase-1 fragments, thus allowing for direct
analysis of caspase-1 activation by various stimuli. When LPS
primed macrophages are stimulated with ATP, the in vitro pro-
cessing rate of caspase-1 and IL-1? is greatly accelerated, and this
phenomenon is strictly dependent on activation of the P2X7R (20).
To verify that LPS is required for efficient activation of caspase-1
by P2X7R stimulation in this system, Bac1 macrophages were
treated according to the schematic illustrated in Fig. 2A. These
macrophages were incubated with or without LPS for 4 h followed
by brief (5 min) stimulation with 1 mM ATP; this 5 min stimula-
tion corresponds to the lag time between the ATP occupation of
P2X7R in intact cells and the initial appearance of activated
caspase-1 in the extracellular medium (Fig. 1B). The cells were
then immediately lysed and caspase-1 processing was monitored in
vitro. Fig. 2B (left end) shows that lysates from control macro-
phages, lacking both LPS priming and acute ATP stimulation,
were characterized by a very low rate of caspase-1 activation. Pre-
vious studies from our group (20) and others (6) have indicated
that this result reflects basal assembly of inflammasome compo-
nents by the hypotonic buffer used to lyse the cells. We previously
reported that this low basal rate is the same in lysates from mac-
rophages that were pretreated with LPS but not stimulated with
ATP (20). In lysates from macrophages primed with LPS and then
stimulated with ATP (Fig. 2B, right end), this processing rate was
greatly accelerated, demonstrating that P2X7R activation in the
presence of LPS priming markedly accelerates assembly of the
caspase-1 activation complexes. In contrast, when macrophages
were not primed by LPS before ATP stimulation, no acceleration
of caspase-1 activation was observed in the corresponding cell-free
lysates (Fig. 2B, middle). This indicates that LPS priming is re-
quired for the rapid intracellular assembly of the caspase-1 acti-
vation machinery in response to P2X7R stimulation independently
of the export of caspase-1.
The activation of caspase-1 by the P2X7R can be mimicked by
treating monocytes or macrophages with other agents, such as
ionophores, that induce K?release from the cell (13, 16, 20, 29).
To determine whether the dependence on LPS priming is specific
7613The Journal of Immunology
to P2X7R or is a feature common to K?release stimuli, cell-free
lysates were prepared from Bac1 macrophages that were stimu-
lated with 10 ?M nigericin for 5 min with or without prior LPS
priming. Fig. 2C illustrates that nigericin and ATP stimulation of
LPS-primed cells yielded cell-free lysates with similarly rapid
rates of in vitro caspase-1 activation; however, in the absence of
LPS priming, nigericin treatment was unable to induce this rapid
activation of caspase-1. This suggests that LPS priming is a gen-
eral prerequisite for caspase-1 activation by stimuli that induce K?
release from inflammatory cells.
by the P2X7R requires priming with
TLR ligands. A, Bac1 macrophages
were pretreated with or without LPS
(500 ng/ml) for 4 h before stimulation
with 1 mM ATP for varying times. Ex-
tracellular medium samples were col-
lected, TCA precipitated, and the pre-
cipitates dissolved in SDS sample
buffer. The cells were lysed directly in
SDS sample buffer. Both fractions
were analyzed via SDS-PAGE and
Western blot using Abs against IL-1?
and caspase-1. B, BALB/c BMDM
were primed with LPS for the indicated
times before 1 mM ATP stimulation
for 30 min as indicated. Extracellular
medium and cell lysates were collected
and processed as in A. Both fractions
were analyzed via SDS-PAGE and
Western blot using Abs against IL-1?
and caspase-1. C, Bac1 macrophages
(left) or primary bone marrow macro-
phages (right) were treated with 1 mM
ATP for 30 min (left) or for the indi-
cated times (right) in the presence or
absence of LPS priming (500 ng/ml for
4 h). The extracellular medium was re-
moved and the cells were lysed in 10%
nitric acid. The K?remaining in the
cells after ATP treatment was then
measured using atomic spectroscopy.
This result is representative of three
separate experiments. D, Bac1 macro-
phages were treated with 500 ng/ml
LPS or 500 ng/ml lipid A for 4 h, then
transferred to BSS and treated with 1
mM ATP for 30 min. The extracellular
medium was collected and extracellu-
lar mIL-1? was measured via ELISA.
The remaining extracellular medium
was precipitated with TCA and extra-
cellular IL-1? was measured (inset) via
SDS-PAGE and Western blot. These
data are indicative of three separate
and/or triplicate experiments.
Activation of caspase-1
7614P2X7R ACTIVATION OF CASPASE-1 REQUIRES ACTIVATION OF NF-?B
PAMPs that target TLRs other than TLR4 can mimic many
responses triggered in macrophages by LPS or lipid A. Because
macrophages express TLR2 and TLR9 in addition to TLR4 (30,
31), Bac1 macrophages were primed for 4 h with PAMP-free con-
trol media, with 10 ?g/ml LPS-free unmethylated CpG DNA 1886
as a TLR9 agonist, with 500 ng/ml LPS as a TLR4 agonist, or with
520 ng/ml 19-kDa lipoprotein from M. tuberculosis as a TLR2
agonist. The primed cells were then stimulated with 1 mM ATP for
5 min followed by lysis and the in vitro processing assay as de-
scribed by Fig. 2A. Fig. 2D demonstrates that the TLR2 ligand
19-kDa lipoprotein was as efficacious as LPS in priming macro-
phages for the activation of caspase-1 by the P2X7R. Although
CpG DNA also facilitated an increased rate of caspase-1 activa-
tion, the response was less robust relative to priming triggered by
other the TLR agonists. This likely reflects a global hyporespon-
siveness of Bac1 macrophages to CpG DNA given that these cells
accumulated less pro-IL-1? relative to that induced by LPS or
LPS-induced potentiation of caspase-1 activation by ATP
requires protein synthesis
A critical downstream consequence of LPS stimulation is the ac-
tivation of the NF-?B pathway, which results in the up-regulated
expression of many proinflammatory gene products (25). Our ob-
servation that 60 min of LPS priming was sufficient for near-max-
imal activation of caspase-1 by P2X7R in intact macrophages (Fig.
1B) suggested that putative coupling or regulatory proteins may be
characterized be rapid rates of synthesis and/or degradation. Fig.
3A demonstrates that a 15 min priming of Bac1 macrophages with
LPS followed by ATP stimulation for 5 min was sufficient to trig-
ger an increased rate of caspase-1 processing in the subsequently
isolated cell-free lysates. However, maximal induction of the
P2X7R-dependent caspase-1 activation was induced by the routine
4 h LPS priming step. This indicates that LPS-mediated priming
may involve a rapidly induced but progressive accumulation of
proteins required for efficient caspase-1 activation by P2X7R
Moreover, pulsing Bac1 cells with LPS for 15 min followed by
extensive washing and further incubation for 3.75 h was also suf-
ficient for maximal potentiation of the caspase-1 activation re-
sponse, suggesting that an early TLR4 signal is sufficient for the
priming activity and that maintained TLR4 activation was not re-
quired. This would be consistent with the up-regulation of a pro-
tein that regulates caspase-1 activation. To determine whether pro-
tein synthesis is required for this LPS-mediated priming response,
Bac1 macrophages were pretreated with cyclohexamide for 30 min
followed by LPS treatment for 4 h. The intact cells were then
pulsed with 1 mM ATP for 5 min followed by lysis and the in vitro
processing assay. Fig. 3B demonstrates that the ability of LPS
priming to facilitate caspase-1 activation by ATP-stimulated
P2X7R is markedly attenuated in the presence of cyclohexamide.
The ability of cyclohexamide to effectively inhibit protein synthe-
sis in these LPS-primed cells was verified by the complete repres-
sion of inducible pro-IL-1? accumulation. However, constitutively
expressed proteins were not affected by the 4 h cyclohexamide
incubation as procaspase-1, and P2X7R levels did not markedly
change with this treatment (see Fig. 4C). As an additional readout
of the relative caspase-1 activities in cell-free lysates from cells
primed with LPS in the absence or presence of cyclohexamide, the
lysates were supplemented with recombinant human IL-1? as a
caspase-1 substrate (Fig. 3C). Lysates from LPS primed, ATP-
stimulated Bac1 macrophages were characterized by a high rate of
recombinant pro-IL-1? cleavage, which was blocked by the
caspase-1 inhibitor YVAD-cmk. Lysates from macrophages incu-
bated with cyclohexamide during the LPS priming and ATP stim-
ulation steps were unable to significantly process the recombinant
Cyclohexamide similarly inhibited the ability of LPS to poten-
tiate P2X7R-mediated caspase-1 activation and export in primary
BMDM (Fig. 4). Incubation of these cells with this protein syn-
thesis inhibitor during the LPS priming did not alter the intracel-
lular levels of procaspase-1, but completely repressed the ATP-
induced accumulation of caspase-1 p10 subunit in the extracellular
medium; this was identical with the results observed when the
BMDM were stimulated with ATP in the absence of LPS priming
(Fig. 4A). If the BMDM were first primed with LPS for 3.5 h in the
absence of cyclohexamide followed by 30 min of cyclohexamide
exposure immediately before the ATP stimulation step, P2X7R-
dependent cleavage and secretion of IL-1? could still be observed
A, Schematic of in vitro processing assay. B, Bac1 macrophages were
pretreated with or without LPS (500 ng/ml) for 4 h and then stimulated
with 1 mM ATP for 5 min. The cells were then lysed in hypotonic buffer
W and subjected to the in vitro processing assay as described in Materials
and Methods. The rate of caspase-1 and IL-1? processing was analyzed via
SDS-PAGE and Western blot. C, Bac1 macrophages were treated with 10
?M nigericin for 5 min in the presence or absence of LPS priming as
described in B. The cells were then lysed in hypotonic buffer W and sub-
jected to the in vitro processing assay described. D, Bac1 macrophages
were primed for 4 h with 10 ?g/ml unmethylated CpG-DNA 1886, 520
ng/ml M. tuberculosis 19-kDa lipoprotein, or 500 ng/ml LPS. The cells
were transferred to BSS and then stimulated with 1 mM ATP for 5 min.
The cells were immediately lysed in a hypotonic buffer, and the lysates
were used in an in vitro processing assay. Processing of IL-1? and
caspase-1 were monitored via SDS-PAGE and Western blot for IL-1? and
caspase-1 p10 Abs. These blots are representative of at least three separate
Activation of caspase-1 by K?loss requires LPS priming.
7615 The Journal of Immunology
but at a reduced level (Fig. 4B); extracellular accumulation of ac-
tive caspase-1 was also reduced by this shorter term of exposure to
cyclohexamide. However, cyclohexamide pretreatment did not
change P2X7R activity as indicated by the K?release assay. Bac1
macrophages primed with LPS for 4 h and then treated with 1 mM
ATP for 5 min released an average of 90 nmol K/well. In the
presence of a 30 min cyclohexamide preincubation followed by an
additional 3.5 h of LPS priming with ATP stimulation as de-
scribed, the macrophages released an average of 84 nmol K/well.
This indicates that the 4 h of cyclohexamide exposure did not alter
the ability of P2X7R to act as ATP-gated cation channels. Collec-
tively, the various data in Figs. 3 and 4 suggest that a putative
coupling protein rapidly accumulates within 15 min of LPS prim-
ing and possesses sufficient stability such that acute blockade of
ongoing protein synthesis (for 30 min) diminishes, but does not
eliminate, the ability of P2X7R to couple to the caspase-1 activa-
LPS priming of P2X7R-mediated caspase-1 activation requires
proteasome activity and NF-?B activation
LPS activation of TLR4 induces MyD88 recruitment to the recep-
tor, followed by IL-1R-associated kinase 1 phosphorylation, and
subsequent recruitment and polyubiquitination of TNFR-associ-
ated factor 6, which itself can act as an E3 ubiquitin ligase. The
proteasome-mediated degradation of several proteins, including
IL-1R-associated kinase 1 and I?B, follows these ubiquitination
for the indicated time points before stimulation with ATP for 5 min. For the 15 min pulse, cells were treated with LPS for 15 min, washed extensively with
PBS, and then the medium was replaced for 4 h before ATP treatment for 5 min. Cells were then lysed and caspase-1 activation was monitored by the in
vitro processing assay. B, Bac1 macrophages were pretreated with 50 ?M cyclohexamide for 30 min followed by 4 h of 500 ng/ml LPS where indicated
in the presence of cyclohexamide. Cells were then bathed in BSS and stimulated with 1 mM ATP for 5 min followed by lysis and the in vitro processing
assay as previously described. The inhibition of protein synthesis by cyclohexamide was determined by Western blot of IL-1?, which is induced by LPS
priming. Caspase-1 activation independent of protein synthesis was determined by Western blot for the p10 subunit of caspase-1. C, Inhibition of caspase-1
activation by pretreatment with cyclohexamide was confirmed using the cleavage of recombinant human IL-1? as a bioassay. COS-1 cells were transfected
with human pro-IL-1? and lysed in hypotonic buffer after 48 h. Bac1 macrophages were primed with LPS or LPS and in the presence of cyclohexamide
as indicated. The cells were then treated with 1 mM ATP for 5 min. The cells were then lysed in hypotonic buffer W and 18.75 ?g of COS-1 lysate
containing recombinant human IL-1? was added to 990 ?g Bac1 lysate. The mixture was then aliquoted and incubated at 30°C for the indicated times.
The reaction was stopped by dilution 1/5 with buffer W and freezing. The processing of IL-1? was monitored via ELISA specific for human mIL-1?. To
ensure that the IL-1? processing reflects caspase-1 activity, lysates were incubated with 10 ?M YVAD before transfer to 30°C. All Western blots were
repeated at least twice. The bioassay is representative of two experiments done in duplicate.
LPS priming of caspase-1 activation by P2X7R is dependent on protein synthesis. A, Bac1 macrophages were primed with 500 ng/ml LPS
7616 P2X7R ACTIVATION OF CASPASE-1 REQUIRES ACTIVATION OF NF-?B
reactions (32). Because protein synthesis is required for LPS prim-
ing of caspase-1 activation by the P2X7R, we tested whether this
protein synthesis occurs via proteasome-sensitive mechanisms and
NF-?B-mediated transcription. Bac1 macrophages were preincu-
bated with the proteasome inhibitor MG132 for 30 min followed
by LPS priming for 3.5 h, pulse stimulation with 1 mM ATP for 5
min before lysis, and analysis by the in vitro processing assay.
Preincubation with MG132 was able to block the priming effect of
LPS on caspase-1 activation by ATP-stimulated P2X7R (Fig. 5A).
However, if the macrophages were treated with LPS for 3.5 h
followed by a 30 min treatment with MG132, the ability of LPS
treatment to potentiate caspase-1 activation by ATP stimulation
was not affected. Treatment of the primary BMDM with MG132
before, but not after, LPS priming similarly repressed P2X7R-de-
pendent processing and release of IL-1? (Fig. 5C). Preincubation
of the cells with MG132 did not affect the ability of activated
P2X7R to induce K?loss (data not shown), nor did it alter levels
of P2X7R protein (Fig. 5D). This indicates the effect of MG132
does not reflect a nonspecific action on cell viability or P2X7R
expression and function. This suggests that for LPS priming to
effectively potentiate caspase-1 activation by the P2X7R, protea-
some function must be intact downstream of the TLR signaling
complex. To verify that MG132 blocked proteasome activity under
the conditions used in the caspase-1 activation experiment, we
assayed the cellular levels of I?B in Bac1 macrophages. Pretreat-
ment with MG132 markedly reduced the rapid degradation of I?B
triggered by acute LPS stimulation (Fig. 5B). Moreover, MG132
completely suppressed the LPS-induced expression of pro-IL-1?
(Fig. 5, A, C, and D), a known NF-?B-dependent gene product.
To further address the possible role of NF-?B signaling in LPS
priming of the P2X7R3caspase-1 activation cascade, we tested
the effects of the IKK inhibitor, Bay 11-7085. Bac1 macrophages
were pretreated with increasing concentrations of Bay 11-7085
during the 4 h LPS priming step before the 30 min ATP pulse (Fig.
6A). Bay 11-7085 produced a concentration-dependent inhibition
of the LPS-dependent expression of pro-IL-1? without affecting
the constitutive expression of procaspase-1. At concentrations ?1
?M, Bay 11-7085 completely repressed P2X7R-dependent activa-
tion of caspase-1 as indicated by the extracellular accumulation of
active caspase-1 p10 subunits. A similar inhibitory action of Bay
11-7085 (at 10 ?M) was observed in primary BMDM (Fig. 6B).
Bay 11-7085 did not affect the ability of ATP to induce K?release
from Bac1 macrophages, indicating that it does not exert direct
inhibitory effects on the P2X7R (Fig. 6C). Likewise, treatment of
primary BMDM with Bay 11-7085 during the 4 h LPS priming
incubation did not change the level of P2X7R protein (Fig. 5D).
Consistent with its action as an IKK inhibitor, Bay 11-7085 also
produced a concentration-dependent repression of the rapid I?B-?
degradation triggered by LPS (Fig. 6D).
LPS potentiation of caspase-1 activation by P2X7R stimulation
does not require the ERK, JNK, p38, or PI3K signaling
Additional downstream signaling pathways induced by TLR acti-
vation include the ERK, JNK, and p38 families of MAPK as well
as PI3K (25, 33). To test whether any of these pathways were
involved in the potentiation of caspase-1 activation by the P2X7R,
Bac1 macrophages were preincubated with various inhibitors for
30 min followed by LPS treatment for 3.5 h. The cells were then
transferred to BSS, treated with 1 mM ATP for 5 min, lysed, and
the lysates were incubated at 30°C for the in vitro processing as-
say. The MEK1 inhibitor U0126, which inhibits the activation of
ERK1 and ERK2 by LPS (Fig. 7B), did not alter the potentiation
dependent on protein synthesis in primary macrophages. A, BALB/c BMDM
were pretreated with or without 50 ?M cyclohexamide for 30 min followed by
1 ?g/ml LPS for an additional 3.5 h; parallel samples of control cells were
neither LPS-primed nor treated with cyclohexamide. Cells were then washed
and treated with ATP for 30 min in BSS followed by isolation of the extra-
cellular medium and cell lysates as in Fig. 1A. B, Cells were treated as in A
(Pre) or the cells were primed with LPS for 3.5 h followed by a 30 min
cyclohexamide addition before washing and stimulation with ATP (Post). The
extracellular medium and cell lysates were isolated and processed as in A. C,
Primary BMDM were treated as in B without ATP stimulation. Whole cell
lysates were analyzed by Western blot for effects of cyclohexamide treatment
on IL-1?, procaspase-1, and P2X7R levels.
LPS priming of caspase-1 activation by P2X7R activation is
7617The Journal of Immunology
of P2X7R-mediated caspase-1 activation by LPS (Fig. 7A). Like-
wise, SP600125, an inhibitor of JNK signaling, produced no effect
on this mode of caspase-1 regulation (Fig. 7A) even when tested at
concentrations that blocked the activation of JNK by anisomycin
in the same cells (Fig. 7C). SB203580, a commonly used inhibitor
of p38 MAPK, did not impact the activation of caspase-1 by ATP
in LPS-primed macrophages but did attenuate the LPS-induced
accumulation of IL-1? (Fig. 7, A and D). Finally, wortmannin, an
inhibitor of PI3K, did not reduce the ability of ATP stimulation to
activate caspase-1 in LPS primed cells (Fig. 7E), even though the
same concentration of this inhibitor blocked the phosphorylation
of its substrate, Akt, in HEK 293 cells challenged with epidermal
growth factor (Fig. 7F). These inhibitor studies indicate that the
ability of LPS priming to potentiate caspase-1 activation by the
P2X7R does not involve an obvious or obligatory role for four of
the kinase pathways responsible for many acute responses to LPS
and other PAMPs. Additionally, because P2X7R activation has
been shown to stimulate each of these four kinase-based signaling
pathways in various cell types, (26, 34–37), these data imply that
these pathways are not required for direct coupling of ATP-gated
P2X7R to the caspase-1 activation machinery.
Caspase-1 is the converting enzyme required for the cleavage and
maturation of both IL-1? and IL-18. Stimulation of LPS-primed
monocytes or macrophages with ATP induces rapid processing
and release of these cytokines. Previous studies by Mehta et al.
(38) showed that if monocytes are not primed with LPS, ATP
treatment cannot induce cleavage of IL-18, which in contrast to
IL-1?, is constitutively expressed. This suggested that the efficient
coupling of P2X7R to the caspase-1 activation machinery requires
one or more signals induced by LPS priming. Two recent reports
have described a similar requirement for priming by TLR ligands
for ATP-induced activation of caspase-1 in murine peritoneal mac-
rophages (4, 5). In this study, we have characterized the LPS-
mediated signaling mechanisms required for this caspase-1 acti-
vation by ATP-gated P2X7R. LPS priming appears to be a general
for 30 min followed by priming with LPS for 4 h. The cells were then bathed in BSS, stimulated with 1 mM ATP for 5 min, lysed, and then subjected
to the in vitro processing assay. The activation of caspase-1 was monitored by Western blot using Abs for IL-1? and the caspase-1 p10 subunit. This blot
is representative of at least three separate experiments. B, Bac1 cells were pretreated with 50 ?M MG132 for 30 min followed by stimulation with 500 ng/ml
LPS for the indicated times before cell lysis. The degradation of I?B was monitored via SDS-PAGE and Western blot. C, Primary BMDM were treated
with 4 h of LPS alone, 50 ?M MG132 for 30 min followed by 3.5 h LPS stimulation (Pre), or LPS for 3.5 h followed by 30 min of 50 ?M MG132 (Post).
Cells were then washed, bathed in BSS, and stimulated with or without 1 mM ATP for 30 min as indicated. Extracellular medium and cell lysates were
processed as in Fig. 1A and analyzed by Western blot analysis. D, Primary BMDM were pretreated with 50 ?M MG132 or 10 ?M Bay 11-7085 for 30
min followed by 3.5 h of LPS stimulation. Whole cell lysates were then analyzed for IL-1?, caspase-1, and P2X7R expression by Western blot analysis.
LPS priming of caspase-1 activation by the P2X7R requires activity of the proteasome. A, Bac1 cells were pretreated with 50 ?M MG132
7618P2X7R ACTIVATION OF CASPASE-1 REQUIRES ACTIVATION OF NF-?B
requirement common to caspase-1 regulation by K?release stim-
uli because activation of caspase-1 by nigericin was similarly de-
pendent on prior treatment of macrophages with LPS. However,
LPS pretreatment of macrophages does not affect the ability of
ATP to activate the P2X7R, as similar K?efflux occurs in re-
sponse to ATP with or without LPS priming. Because we have
previously reported that LPS stimulation of Bac1.2F5 (Bac1) mac-
rophages does not induce ATP release (39), the requirement for
LPS priming does not involve autocrine activation of the P2X7R
by endogenous ligands. In contrast to our findings, another study,
using THP-1 monocytes, suggested that nigericin can induce a
cathepsin B cell-dependent caspase-1 activation in the absence of
LPS priming (40). We have recently reported that THP-1 mono-
cytes have an increased capacity to accumulate active caspase-1,
even in the absence of additional K?release stimuli (20, 41). Thus,
differences between monocyte vs macrophage model systems may
underlie discrepant observations regarding a requirement for LPS
priming for robust activation of caspase-1 by various K?release
We also confirmed that the requisite signals induced by LPS can
be mimicked by E. coli lipid A, M. tuberculosis 19-kDa lipoprotein
and to a lesser extent by CpG DNA, suggesting that common sig-
naling pathways downstream of TLR2, TLR4, and TLR9 are in-
volved in facilitating P2X7R-mediated caspase-1 activation.
Yamamoto et al. (4) have suggested that the adaptors MyD88 and
Toll/IL-1R domain-containing adaptor inducing IFN-? are not re-
quired for this TLR-mediated priming effect, but further studies are
needed to confirm this observation in several model systems.
Recently, the role of long-term (?24 h) LPS stimulation in the
activation of caspase-1 has been questioned. It has been proposed
that MDP, derived from the metabolism of peptidoglycan contam-
inants of LPS, is the key stimulating molecule that induces
blocks the ability of LPS to potentiate ATP-me-
diated caspase-1 activation. Bac1 (A) or BMDM
(B) were treated with the indicated concentra-
tions (A) or 10 ?M (B) of Bay 11-7085 for 30
min before priming with 1 ?g/ml LPS for 3.5 h.
The cells were transferred to BSS and stimulated
with 1 mM ATP for 30 min. The extracellular
medium and cell lysates were isolated and pro-
cessed as in Fig. 1A. C, Bac1 macrophages were
treated with 10 ?M Bay 11-7085 for 30 min fol-
lowed by no LPS (left) or 1 ?g/ml LPS (right)
for 3.5 h. This process was followed by 30 min
of ATP stimulation as indicated. The extracellu-
lar medium was removed and the cells were
lysed in 10% nitric acid. The K?remaining in
the cells after ATP treatment was measured us-
ing atomic spectroscopy. D, BALB/c BMDM
were pretreated with the indicated concentra-
tions of Bay 11-7085 for 30 min followed by 30
min of 500 ng/ml LPS stimulation. The cells
were lysed in sample buffer and analyzed by
SDS-PAGE. The samples were probed using
Abs against I?B-? or actin as a loading control.
The IKK inhibitor, Bay 11-7085,
7619 The Journal of Immunology
inflammasome assembly during prolonged LPS exposure (24).
MDP, however, does not appear to be a factor in the short-term
priming of macrophages for P2X7R-mediated caspase-1 activation
because purified lipid A, which is free of peptide moieties, pro-
duced the same priming effect as LPS (Fig. 1D).
Pretreatment of cells with cyclohexamide before LPS exposure
blocked both the up-regulation of IL-1? expression by LPS and the
ability of LPS to potentiate the activation of caspase-1 by the
P2X7R. Thus, the priming effect of LPS on P2X7R-mediated
caspase-1 activation requires protein synthesis. De novo protein
synthesis involves LPS-induced gene expression, and our results
raised the question as to which transcription factor might be in-
volved. NF-?B is maintained in an inactive state within the cyto-
plasm by association with I?B. Upon stimulation, I?B is phos-
phorylated by IKK, ubiquitinated and degraded by the proteasome
(42). This allows for translocation of NF-?B to the nucleus and
activation of the transcriptional machinery. Preincubation of Bac1
macrophages or primary BMDM with MG132, an inhibitor of the
proteasome, or Bay 11-7085, an IKK inhibitor, before LPS treat-
ment blocked the ability of LPS to potentiate caspase-1 activation
and to trigger rapid degradation of I?B. In contrast, neither MG132
nor Bay 11-7085 affected P2X7R protein levels (Fig. 5D) or
P2X7R-dependent K?efflux (Fig. 6C and data not shown). We
also obtained similar results with the proteasome inhibitor lacta-
cystin (data not shown). This requirement for intact proteasome
and IKK activity for the LPS priming effects suggests that LPS
potentiation of caspase-1 activation by the P2X7R involves acti-
vation of NF-?B-dependent gene transcription.
Previous reports have described additional effects of Bay 11
compounds on the ERK, JNK, and p38 MAPK (43). Ligand oc-
cupancy of TLRs by their cognate PAMPs activates several well-
characterized signaling pathways within monocytes and macro-
phages. LPS activates all three families of the MAPK, and several
reports have implicated PI3K as an important player in mediating
downstream effects of TLR signaling (25, 44, 45). However, our
pharmacological experiments indicate that these conventional sig-
naling pathways activated by TLR4 are not obligatory for the LPS-
dependent priming of caspase-1 activation by the P2X7R (Fig. 7).
Thus, the effects of the Bay 11 compounds in our studies are un-
likely to involve their effects on any of the MAPK pathways. Ad-
ditionally, recent reports have identified RIP2/RICK (receptor-in-
teracting protein 2/RIP-like interacting CLARP kinase) as a target
for SB203580 (46). Because SB203580 treatment does not affect
LPS-mediated potentiation of P2X7R activation of caspase-1, this
suggests that RIP2/RICK is most likely not involved in the LPS-
mediated potentiation process. This is consistent with studies that
used macrophages from RIP2/RICK knockout mice to demonstrate
pretreated with the indicated MAPK inhibitors (10 ?M U0126, 1 ?M SB203580, or 10 ?M SB600125) for 30 min followed by 500 ng/ml LPS stimulation
for 4 h in the presence of the inhibitors. The cells were stimulated with 1 mM ATP for 5 min and then lysed in hypotonic buffer. The lysates were used
in the in vitro processing assay. IL-1? and caspase-1 processing were monitored by Western blot. B, Bac1 macrophages were pretreated with 10 ?M U0126
for 30 min and then stimulated with LPS for 10 min as indicated. The inhibition of ERK phosphorylation by U0126 was verified by Western blot for the
phosphorylated form of ERK (top). Equal amounts of ERK in each lane were also verified by Western blot for ERK (bottom). C, The ability of SP600125
to inhibit c-jun phosphorylation was analyzed by pretreating Bac1 macrophages for 30 min with varying concentrations of SP600125 followed by a 30 min
stimulation with 200 ng/ml anisomycin. The cell lysates were then precipitated with GST-jun followed by an in vitro kinase assay. The phosphorylation
of c-jun was analyzed by Western blot using an Ab specific for the phosphorylated form of c-jun (top). Equal amounts of GST-jun substrate per reaction
were verified by Western blot using a GST Ab. D, Six-well plates of Bac1 macrophages were pretreated with the indicated amounts of SB203580 for 30
min followed by 4 h of stimulation with 500 ng/ml LPS. Cells were lysed in sample buffer and the inhibition of IL-1? up-regulation by SB203580 was
monitored by Western blot using an IL-1? Ab. E, Bac1 macrophages were pretreated with 2 ?M wortmannin for 30 min followed by 500 ng/ml LPS
stimulation for 4 h. The cells were stimulated with 1 mM ATP for 5 min, lysed in hypotonic buffer, and used in the in vitro processing assay described
for A. F, HEK293 cells were pretreated as indicated with 2 ?M wortmannin for 30 min followed by a 100 ng/ml stimulation with epidermal growth factor
for 10 min. Phosphorylation of Akt was measured by SDS-PAGE and Western blot with S473phospho-specific Ab. The membrane was then stripped and
reprobed to verify equal levels of Akt. The inhibitor studies are indicative of three separate and/or triplicate experiments.
LPS priming of caspase-1 activation by the P2X7R does not require the ERK, JNK, p38, or PI3K pathways. A, Bac1 macrophages were
7620 P2X7R ACTIVATION OF CASPASE-1 REQUIRES ACTIVATION OF NF-?B
no requirement for RIP2/RICK in the P2X7R-driven activation of
IL-1? processing (47).
What genes are likely targets for up-regulation and facilitation
of caspase-1 activation? The oligomerization and activation of
caspase-1 is believed to involve assembly of a multiprotein com-
plex, termed the inflammasome. Caspase-1 binds to a scaffold mol-
ecule termed ASC/Pycard, which binds to members of the CAT-
ERPILLER family that contain both a PYRIN domain and a
nucleotide-binding oligomerization (NACHT) domain, including
Nalp1, Nalp2, and Nalp3/cryopyrin (6, 8, 9, 11, 48). ASC is the
critical molecule within this complex because Abs directed against
ASC can disrupt complex formation in vitro (6). In neutrophils and
THP-1 monocytes, ASC expression is increased in response to
LPS treatment (9, 49). Thus, an increased expression of ASC may
overcome interactions with negative regulators such as pyrin, the
target of familial Mediterranean fever (48, 50) and several CARD-
only proteins such as COP, ICEBERG, and CARD-8 (51–53). This
would allow for free ASC to enable efficient assembly of the in-
flammasome following K?release stimuli. In this regard, we have
previously reported that addition of recombinant ASC protein to
macrophage cell-free lysates induces rapid caspase-1 activation
and IL-1? cleavage similar to that induced by P2X7R stimulation
of intact macrophages before cell lysis (20).
In addition to ASC, mouse caspase-11 and human caspase-5 are
both up-regulated by LPS stimulation (54–56). Both of these pro-
teins have been linked to the activation of caspase-1 and are
thought to be functional orthologs (57). Caspase-11 knockout mice
are resistant to LPS-induced death and show decreased serum lev-
els of IL-1? and IL-1? after LPS stimulation (55). Additionally,
caspase-5 has been identified as a component of one of the inflam-
masome subtypes (6). A role for NF-?B in caspase-11 up-regula-
tion is supported by reports that identified a binding site for NF-?B
on the caspase-11 promoter that becomes occupied in response to
LPS (58) and the inhibition of LPS-mediated caspase-11 up-reg-
ulation by wedelolactone, an inhibitor of IKK (59). However, other
groups have reported that caspase-11 up-regulation is sensitive to
pretreatment of cells with the p38 MAPK inhibitor SB203580 (60).
This finding would suggest that caspase-11 is not the critical factor
required for LPS-mediated potentiation of caspase-1 activation by
the P2X7R because SB203580 treatment did not affect caspase-1
activation in response to ATP stimulation (Fig. 7).
Recently, studies have demonstrated that the CATERPILLER
protein Nalp3/cyropyrin in human monocytes is also up-regulated
by inflammatory stimuli. Significantly, within 30 min of LPS stim-
ulation, a 15-fold increase in Nalp3 mRNA was observed (61).
This time frame is consistent with our results showing that a 15
min LPS stimulation was sufficient to prime Bac1 macrophages for
P2X7R-dependent caspase-1 activation. In this experiment, the
cells were treated for 15 min followed by incubation in BSS for 10
min before lysis, so there was sufficient time for Nalp3 to be up-
regulated and allow for caspase-1 activation in response to P2X7R
stimulation. Additionally, other reports have demonstrated that the
early (0.5–2 h) up-regulation of NF-?B is responsible for de-
tectible IL-1? release in mice injected with LPS (62). Thus, the
up-regulation of Nalp3 is an attractive mechanism to explain the
role of TLR-mediated priming in P2X7R activation of caspase-1.
The control of caspase-1 activation and subsequent processing
of IL-1? and IL-18 is critical for the regulation of inflammatory
states in vivo. Understanding how the activation of caspase-1 by
endogenous receptors, such as P2X7, can be modulated by bacte-
rial byproducts may contribute to new therapies for the treatment
of inflammation and more severe conditions, such as septic shock.
These studies show that the activation of caspase-1 by the P2X7R
is dependent on prestimulation of the cells with microbially de-
rived ligands for TLR2, TLR4, or TLR9. This prestimulation does
not require signaling through known TLR pathway kinases such as
ERK, JNK, p38, or PI3K. Additionally, this TLR-mediated poten-
tiation is dependent on protein synthesis and NF-?B function. This
indicates that NF-?B-mediated transcription plays an important
role in the ability of the P2X7R to activate caspase-1. The specific
proteins up-regulated in response to LPS stimulation that allow for
efficient caspase-1 activation by P2X7R stimulation may provide
an important target for treatment of inflammatory diseases.
We especially thank Dr. Aaron A. R. Tobian, Meghan Paninni, and
Nicole Pecorafor generoushelp
Dr. Cathy Carlin, Dr. Richard Eckert, Dr. Fred Heinzel, Dr. Ed Greenfield,
and Dr. Clifford Harding for generous gifts of reagents. We thank
Dr. Ronald Przybylski for critical reading of the manuscript.
and advice. Wealso thank
The authors have no financial conflict of interest.
1. Dinarello, C. A. 1996. Biologic basis for interleukin-1 in disease. Blood 87:
2. Le Feuvre, R. A., D. Brough, Y. Iwakura, K. Takeda, and N. J. Rothwell. 2002.
Priming of macrophages with lipopolysaccharide potentiates P2X7-mediated cell
death via a caspase-1-dependent mechanism, independently of cytokine produc-
tion. J. Biol. Chem. 277: 3210–3218.
3. Rowe, S. J., L. Allen, V. C. Ridger, P. G. Hellewell, and M. K. Whyte. 2002.
Caspase-1-deficient mice have delayed neutrophil apoptosis and a prolonged in-
flammatory response to lipopolysaccharide-induced acute lung injury. J. Immu-
nol. 169: 6401–6407.
4. Yamamoto, M., K. Yaginuma, H. Tsutsui, J. Sagara, X. Guan, E. Seki,
K. Yasuda, S. Akira, K. Nakanishi, T. Noda, and S. Taniguchi. 2004. ASC is
essential for LPS-induced activation of procaspase-1 independently of TLR-as-
sociated signal adaptor molecules. Genes Cells 9: 1055–1067.
5. Mariathasan, S., K. Newton, D. M. Monack, D. Vucic, D. M. French, W. P. Lee,
M. Roose-Girma, S. Erickson, and V. M. Dixit. 2004. Differential activation of
the inflammasome by caspase-1 adaptors ASC and Ipaf. Nature 430: 213–218.
6. Martinon, F., K. Burns, and J. Tschopp. 2002. The inflammasome: a molecular
platform triggering activation of inflammatory caspases and processing of
proIL-?. Mol. Cell 10: 417–426.
7. Poyet, J.-L., S. M. Srinivasula, M. Tnani, M. Razmara, T. Fernandes-Alnemri,
and E. S. Alnemri. 2001. Identification of Ipaf, a human caspase-1-activating
protein related to Apaf-1. J. Biol. Chem. 276: 28309–28313.
8. Srinivasula, S. M., J.-L. Poyet, M. Razmara, P. Datta, Z. Zhang, and
E. S. Alnemri. 2002. The PYRIN-CARD protein ASC is an activating adaptor for
caspase-1. J. Biol. Chem. 277: 21119–21122.
9. Stehlik, C., S. H. Lee, A. Dorfleutner, A. Stassinopoulos, J. Sagara, and
J. C. Reed. 2003. Apoptosis-associated speck-like protein containing a caspase
recruitment domain is a regulator of procaspase-1 activation. J. Immunol. 171:
10. Grenier, J. M., L. Wang, G. A. Manji, W. J. Huang, A. Al-Garawi, R. Kelly,
A. Carlson, S. Merriam, J. M. Lora, M. Briskin, et al. 2002. Functional screening
of five PYPAF family members identifies PYPAF5 as a novel regulator of NF-?B
and caspase-1. FEBS Lett. 530: 73–78.
11. Agostini, L., F. Martinon, K. Burns, M. F. McDermott, P. N. Hawkins, and
J. Tschopp. 2004. NALP3 forms an IL-1?-processing inflammasome with in-
creased activity in Muckle-wells autoinflammatory disorder. Immunity 20:
12. Yamin, T. T., J. M. Ayala, and D. K. Miller. 1996. Activation of the native
45-kDa precursor form of interleukin-1-converting enzyme. J. Biol. Chem. 271:
13. Perregaux, D., and C. A. Gabel. 1994. Interleukin-1? maturation and release in
response to ATP and nigericin: evidence that potassium depletion mediated by
these agents is a necessary and common feature of their activity. J. Biol. Chem.
14. Perregaux, D. G., R. E. Laliberte, and C. A. Gabel. 1996. Human monocyte
interleukin-1? posttranslational processing: evidence of a volume-regulated re-
sponse. J. Biol. Chem. 271: 29830–29838.
15. Perregaux, D. G., and C. A. Gabel. 1998. Human monocyte stimulus-coupled
Am. J. Physiol. 275: C1538–C1547.
16. Cheneval, D., P. Ramage, T. Kastelic, T. Szelestenyi, H. Niggli, R. Hemmig,
M. Bachmann, and A. MacKenzie. 1998. Increased mature interleukin-1? (IL-
1?) secretion from THP-1 cells induced by nigericin is a result of activation of
p45 IL-1?-converting enzyme processing. J. Biol. Chem. 273: 17846–17851.
17. Gudipaty, L., J. Munetz, P. A. Verhoef, and G. R. Dubyak. 2003. Essential role
for Ca2?in the regulation of IL-1? secretion by the P2X7nucleotide receptor in
monocytes, macrophages, and HEK293 fibroblasts. Am. J. Physiol. 285:
7621The Journal of Immunology
18. Walev, I., K. Reske, M. Palmer, A. Valeva, and S. Bhakdi. 1995. Potassium-
inhibited processing of IL-1? in human monocytes. EMBO J. 14: 1607–1614.
19. MacKenzie, A., H. L. Wilson, E. Kiss-Toth, S. K. Dower, R. A. North, and
A. Surprenant. 2001. Rapid secretion of interleukin-1? by microvesicle shedding.
Immunity 15: 825–835.
20. Kahlenberg, J. M., and G. R. Dubyak. 2004. Mechanisms of caspase-1 activation
by P2X7receptor-mediated K?release. Am. J. Physiol. 286: C1100–C1108.
21. Schumann, R. R., C. Belka, D. Reuter, N. Lamping, C. J. Kirschning,
J. R. Weber, and D. Pfeil. 1998. Lipopolysaccharide activates caspase-1 (inter-
leukin-1-converting enzyme) in cultured monocytic and endothelial cells. Blood
22. Grahames, C. B., A. D. Michel, I. P. Chessell, and P. P. Humphrey. 1999. Phar-
macological characterization of ATP- and LPS-induced IL-1? release in human
monocytes. Br. J. Pharmacol. 127: 1915–1921.
23. Perregaux, D. G., P. McNiff, R. Laliberte, M. Conklyn, and C. A. Gabel. 2000.
ATP acts as an agonist to promote stimulus-induced secretion of IL-1? and IL-18
in human blood. J. Immunol. 165: 4615–4623.
24. Martinon, F., L. Agostini, E. Meylan, and J. Tschopp. 2004. Identification of
bacterial muramyl dipeptide as activator of the NALP3/cryopyrin inflammasome.
Curr. Biol. 14: 1929–1934.
25. Martin, M. U., and H. Wesche. 2002. Summary and comparison of the signaling
mechanisms of the Toll/interleukin-1 receptor family. Biochim. Biophys. Acta
26. Humphreys, B. D., J. Rice, S. B. Kertesy, and G. R. Dubyak. 2000. Stress-
activated protein kinase/JNK activation and apoptotic induction by the macro-
phage P2X7 nucleotide receptor. J. Biol. Chem. 275: 26792–26798.
27. Pai, R. K., M. E. Pennini, A. A. Tobian, D. H. Canaday, W. H. Boom, and
C. V. Harding. 2004. Prolonged Toll-like receptor signaling by Mycobacterium
tuberculosis and its 19-kilodalton lipoprotein inhibits ? interferon-induced reg-
ulation of selected genes in macrophages. Infect. Immun. 72: 6603–6614.
28. Laliberte, R. E., J. Eggler, and C. A. Gabel. 1999. ATP treatment of human
monocytes promotes caspase-1 maturation and externalization. J. Biol. Chem.
29. Perregaux, D., J. Barberia, A. J. Lanzetti, K. F. Geoghegan, T. J. Carty, and
C. A. Gabel. 1992. IL-1? maturation: evidence that mature cytokine formation
can be induced specifically by nigericin. J. Immunol. 149: 1294–1303.
30. Noss, E. H., R. K. Pai, T. J. Sellati, J. D. Radolf, J. Belisle, D. T. Golenbock,
W. H. Boom, and C. V. Harding. 2001. Toll-like receptor 2-dependent inhibition
of macrophage class II MHC expression and antigen processing by 19-kDa li-
poprotein of Mycobacterium tuberculosis. J. Immunol. 167: 910–918.
31. Gould, M. P., J. A. Greene, V. Bhoj, J. L. DeVecchio, and F. P. Heinzel. 2004.
Distinct modulatory effects of LPS and CpG on IL-18-dependent IFN-? synthe-
sis. J. Immunol. 172: 1754–1762.
32. Suzuki, N., S. Suzuki, and W. C. Yeh. 2002. IRAK-4 as the central TIR signaling
mediator in innate immunity. Trends Immunol. 23: 503–506.
33. Guha, M., and N. Mackman. 2002. The phosphatidylinositol 3-kinase-Akt path-
way limits lipopolysaccharide activation of signaling pathways and expression of
inflammatory mediators in human monocytic cells. J. Biol. Chem. 277:
34. Budagian, V., E. Bulanova, L. Brovko, Z. Orinska, R. Fayad, R. Paus, and
S. Bulfone-Paus. 2003. Signaling through P2X7 receptor in human T cells in-
volves p56lck, MAP kinases, and transcription factors AP-1 and NF-?B. J. Biol.
Chem. 278: 1549–1560.
35. Donnelly-Roberts, D. L., M. T. Namovic, C. R. Faltynek, and M. F. Jarvis. 2003.
Mitogen-activated protein kinase and caspase signaling pathways are required for
P2X7receptor (P2X7R)-induced pore formation in human THP-1 cells. J. Phar-
macol. Exp. Ther. 308: 1053–1061.
36. Gendron, F.-P., J. T. Neary, P. M. Theiss, G. Y. Sun, F. A. Gonzalez, and
G. A. Weisman. 2003. Mechanisms of P2X7receptor-mediated ERK1/2 phos-
phorylation in human astrocytoma cells. Am. J. Physiol. 284: C571–C581.
37. Jacques-Silva, M. C., R. Rodnight, G. Lenz, Z. Liao, Q. Kong, M. Tran, Y. Kang,
F. A. Gonzalez, G. A. Weisman, and J. T. Neary. 2004. P2X7receptors stimulate
AKT phosphorylation in astrocytes. Br. J. Pharmacol. 141: 1106–1117.
38. Mehta, V. B., J. Hart, and M. D. Wewers. 2001. ATP-stimulated release of in-
terleukin (IL)-1? and IL-18 requires priming by lipopolysaccharide and is inde-
pendent of caspase-1 cleavage. J. Biol. Chem. 276: 3820–3826.
39. Beigi, R. D., and G. R. Dubyak. 2000. Endotoxin activation of macrophages does
not induce ATP release and autocrine stimulation of P2 nucleotide receptors.
J. Immunol. 165: 7189–7198.
40. Hentze, H., X. Y. Lin, M. S. Choi, and A. G. Porter. 2003. Critical role for
cathepsin B in mediating caspase-1-dependent interleukin-18 maturation and
caspase-1-independent necrosis triggered by the microbial toxin nigericin. Cell
Death Differ. 10: 956–968.
41. Kahlenberg, J. M., and G. R. Dubyak. 2004. Differing caspase-1 activation states
in monocyte versus macrophage models of IL-1? processing and release. J. Leu-
kocyte Biol. 76: 676–684.
42. Silverman, N., and T. Maniatis. 2001. NF-?B signaling pathways in mammalian
and insect innate immunity. Genes Dev. 15: 2321–2342.
43. Relic ´, B., V. Benoit, N. Franchimont, C. Ribbens, M.-J. Kaiser, P. Gillet, M.-P.
Merville, V. Bours, and M. G. Malaise. 2004. 15-deoxy-?12,14-prostaglandin J2
inhibits Bay 11-7085-induced sustained extracellular signal-regulated kinase
phosphorylation and apoptosis in human articular chondrocytes and synovial fi-
broblasts. J. Biol. Chem. 279: 22399–22403.
44. Hora ´k, J. 2003. The role of ubiquitin in down-regulation and intracellular sorting
of membrane proteins: insights from yeast. Biochim. Biophys. Acta 1614:
45. Lee, J. Y., J. Ye, Z. Gao, H. S. Youn, W. H. Lee, L. Zhao, N. Sizemore, and
D. H. Hwang. 2003. Reciprocal modulation of Toll-like receptor-4 signaling
pathways involving MyD88 and phosphatidylinositol 3-kinase/AKT by saturated
and polyunsaturated fatty acids. J. Biol. Chem. 278: 37041–37051.
46. Argast, G. M., N. Fausto, and J. S. Campbell. 2005. Inhibition of RIP2/RIck/
CARDIAK activity by pyridinyl imidazole inhibitors of p38 MAPK. Mol. Cell
Biochem. 268: 129–140.
47. Kobayashi, K., N. Inohara, L. D. Hernandez, J. E. Galan, G. Nunez,
C. A. Janeway, R. Medzhitov, and R. A. Flavell. 2002. RICK/Rip2/CARDIAK
mediates signalling for receptors of the innate and adaptive immune systems.
Nature 416: 194–199.
48. Chae, J. J., H. D. Komarow, J. Cheng, G. Wood, N. Raben, P. P. Liu, and
D. L. Kastner. 2003. Targeted disruption of pyrin, the FMF protein, causes
heightened sensitivity to endotoxin and a defect in macrophage apoptosis. Mol.
Cell 11: 591–604.
49. Shiohara, M., S. Taniguchi, J. Masumoto, K. Yasui, K. Koike, A. Komiyama, and
J. Sagara. 2002. ASC, which is composed of a PYD and a CARD, is up-regulated
by inflammation and apoptosis in human neutrophils. Biochem. Biophys. Res.
Commun. 293: 1314–1318.
50. Dowds, T. A., J. Masumoto, F. F. Chen, Y. Ogura, N. Inohara, and G. Nunez.
2003. Regulation of cryopyrin/Pypaf1 signaling by pyrin, the familial Mediter-
ranean fever gene product. Biochem. Biophys. Res. Commun. 302: 575–580.
51. Humke, E. W., S. K. Shriver, M. A. Starovasnik, W. J. Fairbrother, and
V. M. Dixit. 2000. ICEBERG: a novel inhibitor of interleukin-1? generation. Cell
52. Lee, S. H., C. Stehlik, and J. C. Reed. 2001. COP, a caspase recruitment domain-
containing protein and inhibitor of caspase-1 activation processing. J. Biol. Chem.
53. Razmara, M., S. M. Srinivasula, L. Wang, J.-L. Poyet, B. J. Geddes,
P. S. DiStefano, J. Bertin, and E. S. Alnemri. 2002. CARD-8 protein, a new
CARD family member that regulates caspase-1 activation and apoptosis. J. Biol.
Chem. 277: 13952–13958.
54. Campos, S. P., Y. Wang, and H. Baumann. 1996. Insulin modulates STAT3
protein activation and gene transcription in hepatic cells. J. Biol. Chem. 271:
55. Wang, S., M. Miura, Y. K. Jung, H. Zhu, E. Li, and J. Yuan. 1998. Murine
caspase-11, an ICE-interacting protease, is essential for the activation of ICE.
Cell 92: 501–509.
56. Lin, X. Y., M. S. Choi, and A. G. Porter. 2000. Expression analysis of the human
caspase-1 subfamily reveals specific regulation of the CASP5 gene by lipopoly-
saccharide and interferon-?. J. Biol. Chem. 275: 39920–39926.
57. Van de Craen, M., P. Vandenabeele, W. Declercq, I. Van den Brande,
G. Van Loo, F. Molemans, P. Schotte, W. Van Criekinge, R. Beyaert, and
W. Fiers. 1997. Characterization of seven murine caspase family members. FEBS
Lett. 403: 61–69.
58. Schauvliege, R., J. Vanrobaeys, P. Schotte, and R. Beyaert. 2002. Caspase-11
gene expression in response to lipopolysaccharide and interferon-? requires nu-
clear factor-?B and signal transducer and activator of transcription (STAT)1.
J. Biol. Chem. 277: 41624–41630.
59. Kobori, M., Z. Yang, D. Gong, V. Heissmeyer, H. Zhu, Y. K. Jung,
M. A. Gakidis, A. Rao, T. Sekine, F. Ikegami, et al. 2004. Wedelolactone sup-
presses LPS-induced caspase-11 expression by directly inhibiting the IKK com-
plex. Cell Death Differ. 11: 123–130.
60. Hur, J., S. Y. Kim, H. Kim, S. Cha, M. S. Lee, and K. Suk. 2001. Induction of
caspase-11 by inflammatory stimuli in rat astrocytes: lipopolysaccharide induc-
tion through p38 mitogen-activated protein kinase pathway. FEBS Lett. 507:
61. O’Connor, W., Jr., J. A. Harton, X. Zhu, M. W. Linhoff, and J. P. Ting. 2003.
Cutting edge: CIAS1/cryopyrin/PYPAF1/NALP3/CATERPILLER 1.1 is an in-
ducible inflammatory mediator with NF-?B suppressive properties. J. Immunol.
62. Han, S.-J., H.-M. Ko, J.-H. Choi, K. H. Seo, H.-S. Lee, E.-K. Choi, I.-W. Choi,
H.-K. Lee, and S.-Y. Im. 2002. Molecular mechanisms for lipopolysaccharide-
induced biphasic activation of nuclear factor-?B (NF-?B). J. Biol. Chem. 277:
7622P2X7R ACTIVATION OF CASPASE-1 REQUIRES ACTIVATION OF NF-?B