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INTRODUCTION |
IL-1
1 and IL-18 are
proinflammatory cytokines that require processing by the
IL-1-converting enzyme, caspase-1, at specific aspartic acid residues
to generate functional molecules (1-3). Although lacking in sequence
homology, IL-1
and IL-18 share significant structural homology (4,
5), bind to receptors from the IL-1 family of receptors (6), exist in
the cytosol without a classic signal sequence (3, 7), and are released
from cells in a noncanonical fashion (8). Both molecules are regulated
posttranscriptionally, and a particularly important aspect of
regulation occurs at the level of release (9-14). In this context,
recent studies show that this release pathway can be rapidly induced by
stimulation with exogenous ATP (15-18). In macrophages, exogenous ATP
works via the recently cloned P2X7 receptor, a member of
the P2X family of nucleotide-gated channels that are activated by
extracellular ATP (19). However, it has been suggested that monocytes
do not express P2X7 receptors (20, 21). P2X7 is
a 595-amino acid polypeptide with two membrane-spanning domains and
intracellular N- and C-terminal domains (22, 23). P2X7
receptor is the only pore-forming P2X family member, and its activation
results in the opening of a cationic channel with increased
permeability to calcium and intracellular depolarization (20). The
present work focuses on release mechanisms by taking advantage of the dramatic ability of ATP to telescope the processing and release down to
a 15-30-min interval while enhancing the overall release signal
(15).
In this report we examine the mechanism of ATP-induced release of IL-18
and compare it to IL-1
in human monocytes. We show that fresh
monocytes express functional P2X7 receptors that modulate IL-1
and IL-18 processing and release in response to ATP. Of note,
our data suggest that ATP stimulation alone is insufficient to trigger
IL-18 processing and release, since priming with LPS was shown to be
necessary. Additionally, the ATP effect on release does not require
functional caspase-1, confirming for IL-18 what has been previously
shown for IL-1
, i.e. precursor cleavage is not required
for release. Finally, attempts to characterize the signaling pathway
involved in the ATP effect show that a protein-tyrosine kinase
inhibitor, AG126, can potently block activation of caspase-1 and
IL-1
/IL-18 release events, whereas mitogen-activated protein kinase,
PKC and PKA inhibitors do not.
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MATERIALS AND METHODS |
Reagents--
ProIL-1
-specific rabbit polyclonal antibody
(amino acids, 3-21) was developed in our laboratory. Human recombinant
IL-1
, biotinylated IL-18, anti-human monoclonal IL-1
antibody
(clone 8516), and IL-18 ELISA kit were purchased from R&D Systems
(Minneapolis, MN). Mature IL-1
and precursor IL-1
-specific ELISA
were from our own laboratory. Polyclonal anti-human P2X7
antibody was from Alamone Laboratories (Jerusalem, Israel), and
monoclonal antibody to P2X7 was a gift from Dr. Ian
Chessell (University of Cambridge, Cambridge, UK). Recombinant
caspase-1 (ICE) and caspase-1 antibody were gifts from Dr. Nancy
Thornberry and Dr. Doug Miller (Merck). Herbimycin A, caspase-1
inhibitor (Ac-Tyr-Val-Ala-Asp-chloromethyl ketone (YVAD-cmk)), and
tyrphostins and other protein kinase inhibitors were obtained from
Calbiochem. Bacterial lipopolysaccharide, Escherichia coli
strain 0127:B8, Westphal preparation, was from Difco. LNAME was from
Biomol (Plymouth Meeting, PA). Phospho-ERK antibody was obtained from
New England Biolab (Plymouth Meeting, PA), and total ERK antibody was
from Upstate Biotechnology Inc. (Lake Placid, NY). RPMI 1640 and
phosphate-buffered saline were purchased from Biowhittaker Inc.
(Walkersville, MD), and fetal bovine serum was from Hyclone
Laboratories (Logan, UT). All other reagents were obtained from Sigma
unless otherwise specified.
Isolation of Human Peripheral Blood Monocytes and Cell
Culture--
Human peripheral blood monocytes were isolated from the
heparinized blood of normal donor using histopaque density gradient followed by a monocyte clumping method as described (24). This method
yields about 65-75% pure monocytes. Isolated monocytes were cultured
at 106/ml in RPMI 1640 supplemented with 5% fetal bovine
serum at 37 °C in humidified incubator. Some cells were cultured for
3 h in the presence of LPS (1 ng/ml) and in the presence or
absence of caspase-1 (ICE) inhibitor YVAD-cmk (100 µM).
Preparation of Cell Lysates and Western Blot Analysis--
Where
indicated, LPS-primed human monocytes were challenged with 5 mM ATP and incubated for an additional 30 min. Culture media was removed from the cells and centrifuged at 800 × g for 5 min, and the supernatants and cell pellets were
collected. Cells were lysed in lysis buffer (50 mM Tris-Cl,
pH 8.0, 150 mM NaCl, 2 mM EDTA, and 1% Nonidet
P-40) containing 2 µg/ml leupeptin, aprotinin, chymostatin,
pepstatin, antipain, 0.5 mM phenylmethylsulfonyl fluoride,
and 50 µM N-methoxysuccinyl-Ala-Ala-Pro-Val
chloromethyl ketone. The cell debris and nuclei were removed by
centrifugation at 14,000 × g for 20 min. For the
analysis of phospho-ERK in cell lysates of monocytes, 1 mM
Na3VO4 and 50 mM NaF were included in lysis buffer. The protein concentration in cell lysates was determined using the Bio-Rad protein assay reagent. The proteins were
resolved by SDS-PAGE and transferred to nitrocellulose membrane. Nonspecific sites on nitrocellulose membrane were blocked with 5%
nonfat dry milk (Carnation) in TBST (25 mM Tris-Cl, pH-7.5, 150 mM NaCl, 0.05% Tween 20) for 3 h at room
temperature. For immunoblotting with phospho-ERK antibody, the membrane
was blocked with 3% bovine serum albumin in TBST. The membranes were
then probed with primary antibodies as indicated followed by
peroxidase-conjugated secondary antibodies, and protein bands were
visualized by enhanced chemiluminescence (ECL, Amersham Pharmacia Biotech).
Flow Cytometric Analysis of P2X7
Receptors--
Isolated monocytes were cultured in complete media
alone or in complete media containing LPS (1 ng/ml) for 3 h or
overnight in LPS (1 µg/ml). For flow cytometric analysis, cells were
stained with monoclonal antibody to P2X7 essentially as
described (25). Briefly, cells were pelleted, washed twice with wash
buffer (1% bovine serum in phosphate-buffered saline), and incubated
in wash buffer containing 4 µg/ml monoclonal P2X7 or an
isotype (IgG2b) control antibody for 30 min at 4 °C
followed by fluorescein isothiocyanate-labeled or rhodamine-labeled rat
anti-mouse IgG-specific F(ab')2 fragments (Jackson
Immunoresearch laboratories, Westgrove, PA). Some cells were also
stained for CD14. For the analysis of P2X7 receptor expression, monocytes were gated for CD14 fluorescence. Flow cytometric analysis was performed using Becton Dickinson FACS Calibur.
IL-1
and IL-18 ELISA--
Sandwich ELISAs were developed in
our laboratory to detect pro- and mature IL-1
as described (13). The
coating antibody has been modified since the previous description.
Briefly, anti-human mouse monoclonal IL-1
antibody (clone 8516, R&D
Systems) was used as a coating antibody, and rabbit polyclonal
proIL-1
-specific peptide antibody generated against amino acids
3-21 was used to sandwich the antigen. Horseradish peroxidase
(Bio-Rad)-conjugated goat anti-rabbit antibody was used as a developing
antibody. The mature IL-1
ELISA used monoclonal antibody clone 8516 and a rabbit polyclonal mature IL-1
antibody (raised against entire
17-kDa mature IL-1
) as coating and sandwich antibodies,
respectively. For the measurement of IL-18, ELISA kits were purchased
from R&D Systems, and cell associated and released IL-18 contents were determined per manufacturer instructions.
Statistical Analysis--
Data are presented as the means ± S.E. of the mean from at least three independent experiments. Simple
comparisons were done by paired t test, with
p < 0.05 considered to represent statistical significance.
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RESULTS |
ATP Promotes IL-1
Release--
To establish the optimum timing
for future analyses of IL-1
processing, we initially examined the
kinetics of ATP-induced IL-1
release from LPS-primed human
peripheral blood monocytes (Fig. 1).
After 3 h of LPS (1 ng/ml), monocytes contained high levels of
intracellular IL-1
with only minimal release. However, ATP (5 mM) induced a dramatic release of mature IL-1
, most
pronounced between 15-20 min, that began to level off after 30 min of
ATP. The accumulation of IL-1
in culture medium correlated with a decrease in cell-associated IL-1
. In all subsequent experiments, ATP
treatment was standardized at 30 min.

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Fig. 1.
Kinetics of IL-1 release from LPS-primed
peripheral blood monocytes after ATP challenge. Human blood
monocytes from a normal donor were cultured at a density of
106 cells/ml in 24-well plates. Cells were primed with LPS
(1 ng/ml) and incubated at 37 °C for 3 h. ATP (5 mM) was then added to the cells, and at indicated times,
cells and supernatants were collected. Cell-associated and -released
IL-1 s were measured by proIL-1 -specific and mature sandwich
ELISA, respectively.
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ATP-stimulated Release of IL-1
and IL-18 Requires LPS
Priming--
Having established the kinetics of the ATP effect on
IL-1
, we next compared the related cytokine IL-18 to IL-1
for
ATP-mediated release. As shown in Fig. 2,
A and B, the LPS priming induced a dramatic release of both IL-1
and
IL-18 at 30 min after ATP treatment. However, there were significant
differences in the ability of LPS to induce IL-1
compared with
IL-18. Whereas unprimed monocytes contained very little IL-1
intracellularly (Fig. 2C) (2.2 ± 1.5 ng/106 cells), low dose LPS induced a dramatic induction of
intracellular proIL-1
(30.2 ± 12.1 ng/106 cells).
In contrast, there was no difference between unprimed and LPS-primed
monocytes for the presence of intracellular IL-18 (Fig. 2D)
(378 ± 22 versus 409 ± 5 pg/106
cells, p = not significant).

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Fig. 2.
ATP-stimulated release of IL-18 requires
priming by LPS. Monocytes were plated in the presence or absence
of LPS (1 ng/ml) for 3 h at 37 °C. Cells were then exposed to 5 mM ATP for 30 min. Released (A and B)
and cell-associated (C and D) IL-1 and IL-18
were determined by sandwich ELISA as described. The data represent the
mean ± S.E. of three independent experiments performed in
duplicate.
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The constitutive presence of IL-18 in unprimed monocytes provided a
unique opportunity to determine whether monocyte IL-18 processing and
release induced by ATP requires a priming signal. Of particular note,
despite the constitutive presence of intracellular IL-18 in the
unprimed monocytes, ATP alone had no effect on inducing its release
(Fig. 2B) (control, 54 ± 19 versus ATP,
67 ± 28 pg/106 cells). However, after priming with
LPS, ATP-induced IL-18 release increased 4-fold (215 ± 60 pg/106 cells; p < 0.05 compared with
control). Our data thus demonstrate that ATP-stimulated release of
IL-18 requires priming by LPS.
Caspase-1 Inhibition Does Not Prevent the Release of IL-1
and
IL-18--
Caspase-1, which is critical for processing IL-1
and
IL-18, has been variably linked to the release pathway for these
cytokines. It has been shown for example that caspase-1
/
macrophages are impaired for IL-1
release (26). To investigate the
requirement for caspase-1 function in ATP-induced IL-1
and IL-18
release, we inhibited caspase-1 with the tetrapeptide, YVAD-cmk (100 µM). We analyzed IL-1
and IL-18 release with ELISAs
able to detect both cytokines in the precursor form (13). Human
monocytes were cultured with YVAD and LPS in varying combinations (Fig.
3). As we and others have previously
shown (13, 27), the presence of an ICE inhibitor prevented mature
IL-1
release from LPS-primed, ATP-stimulated, monocytes (data not
shown). Importantly, however, a significant quantity of proIL-1
was
detected in supernatants using a proIL-1
-specific ELISA (Fig. 3,
A and C).

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Fig. 3.
Processing of IL-1
and IL-18 is not required for ATP-induced release. Monocytes
were cultured in the presence of either Me2SO
(DMSO) alone, YVAD (100 µM) alone, or LPS and YVAD
combined. Where indicated, after 3 h cells were challenged with
ATP (5 mM) for 30 min. Samples were analyzed with ELISAs
that detect the cytokines in their precursor forms. IL-1 . Released
(A) and cell-associated (C) IL-1 and IL-18
were measured by the proIL-1 -specific ELISA. Released (B)
and cell associated (D) IL-18 was measured by R&D Systems
IL-18 ELISA.
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As was shown for IL-1
, after LPS priming, ATP induced monocytes to
release IL-18 (Fig. 3, B and D). Although ATP
alone did not stimulate the release of IL-18 from cells treated with
caspase-1 inhibitor, ICE inhibition did not prevent IL-18 release from
LPS-primed, ATP-stimulated monocytes (Fig. 3, B and
D). These data suggest that release of IL-1
and IL-18
does not require active ICE. Therefore, as has been shown for IL-1
,
the processing of IL-18 is not required for its externalization.
ATP Treatment Promotes IL-1
and IL-18 Processing--
To
confirm that the ATP treatment not only induces release but also
promotes processing of IL-1
and IL-18, monocyte samples were also
assayed by immunoblots (Figs. 4,
A and B). We assessed the effects of ATP on the
processing of IL-18 and IL-1
in untreated monocytes or in monocytes
treated with YVAD or LPS alone or combined LPS and YVAD. Only the
24-kDa precursor form of IL-18 and the 31-kDa form of IL-1
were
detectable in cell lysates. In cell supernatants of unstimulated or
LPS-primed cells, no IL-1
was detected; however, low levels of the
precursor form of IL-18 were detectable. In LPS-primed, ATP-stimulated
monocyte supernatants, a large amount of 18-kDa IL-18 was detected. In
the same supernatants, the 31-kDa form of proIL-1
was not observed,
but a large amount of mature 17-kDa IL-1
was detected.

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Fig. 4.
ATP stimulates processing and release of
IL-1 and IL-18. Monocytes were stimulated
with LPS (1 ng/ml) in the presence or absence of 100 µM
YVAD for 3 h and challenged with ATP for 30 min. Lysates and
supernatants were analyzed by 10-20% gradient SDS-PAGE for IL-1
(A) or 15% SDS-PAGE for IL-18 (B). The proteins
were transferred to nitrocellulose and blotted with
anti-proIL-1 -specific or biotinylated IL-18 antibodies. In the
lower panel of A the blot was stripped and
reprobed with anti-mature IL-1 antibody. rIL-1 ,
recombinant IL-1 .
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In the presence of YVAD-cmk, only precursor forms of IL-18 and IL-1
were detected in supernatants of LPS-primed, ATP-treated monocytes
(Fig. 4, A and B). There was no obvious
difference in the amount of secreted IL-18 and IL-1
in the presence
or absence of YVAD. These observations show that YVAD blocks maturation
of proIL-1
and IL-18 but does not block the release. Thus, both IL-18 and IL-1
require functional caspase-1 for processing during export.
ATP Induces ICE Activation--
It has been reported that
prolonged ATP treatment (3 h) activates caspase-1 in LPS-primed human
monocytes (28). It is not clear whether the short exposure to ATP (30 min) in our experimental system also activates ICE in LPS-primed human
monocytes. Therefore, we examined the effect of ATP and YVAD on
activation of ICE by immunoblot analysis (Fig.
5A). Monocytes were cultured
in the presence or absence of LPS and with or without YVAD for 3 h. ATP (5 mM) was then added where indicated, and cells
were incubated for an additional 30 min. In control and LPS-primed
cells, the 45-kDa precursor and intermediate forms of ICE were
detected, but very little 20-kDa (active form of ICE) was seen.
However, LPS-primed monocytes treated with ATP generated a
significantly higher level of mature subunits (p20 and p10) of ICE,
which was blocked by the presence of YVAD-cmk.

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Fig. 5.
ATP induces activation of caspase-1.
A, cells were cultured either alone or in the presence of
LPS (1 ng/ml) and with or without YVAD-cmk (100 µM) for
3 h, and then ATP (5 mM) was added where indicated.
Lysates were resolved on 10-20% gradient SDS-PAGE, transferred to
polyvinylidene difluoride (Amersham Pharmacia Biotech) membrane, and
probed with polyclonal anti-caspase-1 (ICE) antibody. rICE,
recombinant ICE. B, to show the effect of ATP alone, the
experiment in A was repeated with the inclusion of ATP
without LPS.
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Since, in the absence of LPS priming, ATP alone had no effect on IL-18
processing and release, it was important to determine whether ATP alone
could activate ICE. As shown in Fig. 5B, ATP without LPS
activated ICE to the p20 form in both LPS-primed and unprimed
monocytes. Thus, the data from ELISA and immunoblots, taken together
with the evidence of ICE activation, show that ATP not only activates
ICE but also the processing and release of IL-1
and IL-18. Although
YVAD-cmk blocks the activation of ICE and the maturation of these
precursor cytokines, it fails to inhibit the release of proIL-1
and
proIL-18. Conversely, in the absence of LPS priming, ATP-induced ICE
activation does not induce processing and release. Thus, although ATP
induces ICE activation, it requires an additional signal to induce
processing and release of IL-1
and IL-18.
Monocyte Expression of P2X7 Receptors--
A number of
reports to date suggest that ATP-induced release of IL-1
in
LPS-primed monocytes occurs via P2X7 receptors (15, 25,
29). Human monocytes cultured for 1-3 days in the presence of LPS or
monocytes cultured overnight in the absence of any stimulus contain
P2X7 receptors (25). However, it is not yet clear if fresh
human monocytes cultured for 3 h with or without LPS express P2X7 receptors. To address this question, we analyzed human
monocytes cultured in the presence or absence of LPS for 3 h for
the expression of P2X7 receptors by flow cytometry and
immunoblot analysis. By flow cytometry, a significant signal for
P2X7 receptor was observed in human monocytes when compared
with the isotype control. Importantly, the reactivity with
P2X7 antibody was only minimally enhanced in cells cultured
in the presence of LPS for 3 h (Fig.
6). Immunoblot analysis confirmed the
results of flow cytometry. A 55-kDa band was detected in LPS-stimulated
and unstimulated monocyte lysates that was competed away with the
immunizing peptide (Fig. 7). These data
confirm that fresh monocytes express P2X7 receptors that are only minimally increased by LPS.

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Fig. 6.
Characterization of P2X7
receptors on human blood monocytes. Human monocytes were cultured
alone or with LPS (1 ng/ml) for 3 h. Cells were washed with PBS
and incubated on ice with 4 µg/ml monoclonal anti-P2X7
antibody for 30 min. The P2X7 receptors were detected with
a fluorescein isothiocyanate-labeled donkey anti-mouse
F(ab')2 fragment. An IgG2b antibody was used an
isotype control. The monocytes were gated by anti-CD14 antibody
fluorescence for the presence of P2X7 receptors by flow
cytometric analysis.
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Fig. 7.
Immunoblot of P2X7 receptor
expression on human monocytes. Cells lysates from LPS-primed or
unprimed monocytes were subjected to 10-20% gradient SDS-PAGE,
transferred to nitrocellulose, and probed with polyclonal
anti-P2X7 receptor antibody (Alamone Laboratories)
as described. A, blot probed with P2X7 receptor
antibody; B, parallel blot probed with anti-P2X7
antibody containing immunizing peptide (1:1 w/w). Lysate of rat brain
(R.B.) membrane showing 56- and 80-kDa P2X7 was
used as positive control. hP2X7, human
P2X7.
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Oxidized ATP Blocks the ATP-stimulated Release of IL-1
in
LPS-primed Human Monocytes--
We then asked the question whether
monocytes express functional P2X7 receptors. Therefore, we
attempted to determine if the P2X7 receptor antagonist,
oxidized ATP, and a P2X7-specific antibody prevents the
ATP-stimulated release of IL-1
in LPS-primed human monocytes.
Pretreatment with 300 µM oxidized ATP for 30 min
completely abolished the ATP-induced release of IL-1
in human
monocytes (Fig. 8). Pretreatment with
P2X7-specific monoclonal antibody also blocked the
ATP-stimulated release of IL-1
but to a lesser extent (Fig. 8). The
data suggest that the P2X7 receptor is responsible for the
ATP effects.

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Fig. 8.
Effect of oxidized ATP and P2X7
blocking antibody on ATP-induced release of IL-1
from human monocytes. Monocytes were cultured alone or with
LPS for 3 h. ATP was added to selected samples for the final 30 min. Where indicated, P2X7 monoclonal antibody
(P2X7ab) was given with the LPS, and oxidized ATP
(oATP) was added 30 min before ATP. Released (A)
IL-1 contents were measured by mature IL-1 ELISA, and
cell-associated (B) was measured by proIL-1 -specific
ELISA. Results from duplicate experiments are shown.
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Effect of Signaling Inhibitors on ATP-stimulated Release of
IL-1
--
We hypothesized that protein kinases may play a
significant role in the release of IL-18 and IL-1
from LPS-primed
human monocytes. Initially, we compared the effects of various
inhibitors of protein-tyrosine kinases, protein kinase C, and protein
kinase A on release of IL-1
from LPS-primed human monocytes (Table
I). Whereas platelet-derived growth factor (AG1296) and epidermal growth factor (AG1478) receptor kinase inhibitors and PKA or PKC pathway inhibitors had no effect, tyrphostin AG126, a PTK inhibitor, prevented ATP-induced release of
IL-1
from LPS-primed monocytes (~80% inhibition). The effect was
dose-dependent and not cytotoxic to the cells in the
concentrations used in the experiment. It therefore appears that
ATP-stimulated IL-1
release is mediated by an AG126-sensitive
tyrosine kinase(s).
MEK Inhibitors Do Not Block IL-1
Release--
AG126 is a potent
inhibitor of PTK and inhibits NO production and ERK activation while
protecting mice from LPS-induced septic shock (30, 31). Therefore, we
asked whether the AG126 effect on monocyte IL-1
and IL-18 release is
due to the block in NO production or inhibition of ERK activation.
Using ELISA detection, we first assessed the effects of PTK inhibitors
herbimycin A and AG126, mitogen-activated protein kinase kinase
inhibitors UO126 and PD98059, and nitric-oxide synthase inhibitor LNAME
on ATP-induced release of IL-1
in LPS-primed monocytes (Fig.
9A). Again, AG126 inhibited
IL-1
release in a dose-dependent manner, whereas the
other PTK inhibitor herbimycin A and the MEK inhibitors UO126 and
PD98059 did not inhibit ATP induced IL-1
release (Fig.
9A).

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Fig. 9.
AG126 blocks ATP-stimulated release of
IL-1 but not ERK activation.
LPS-activated cells were preincubated with various inhibitors for 15 min and then exposed to 5 mM ATP for 30 min. A,
released IL-1 was analyzed in cell supernatants using the mature
IL-1 ELISA. The bar graph represents the average ± S.E. of three independent experiments performed in duplicate.
Inhibitors include PD98059 (PD) and herbimycin A
(HA). DMSO, Me2SO. B, cell
lysates were analyzed by blotting with polyclonal anti-phospho-ERK
antibody (top). The blot was stripped and reprobed with
total ERK antibody (bottom), and proteins were visualized
by ECL. The p42 and p44 forms of ERK are abbreviated as
p42ERK and p44ERK, whereas the corresponding
phosphorylated ERK forms are abbreviated as pp42ERK and
pp44ERK.
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To examine whether the AG126 effect was due to the inhibition of NO
production, we analyzed ATP-induced IL-1
release in the presence of
LNAME, a nitric-oxide synthase inhibitor. Interestingly, instead of the
inhibition of release, LNAME enhanced IL-1
release by 2-3-fold. Of
note, AG126 totally abolished the LNAME activated release of IL-1
(Fig. 9A).
Effect of AG126 on ERK Phosphorylation--
We then evaluated
whether AG126 inhibits ATP-induced ERK phosphorylation (Fig.
9B). Whereas the classic MEK inhibitors, UO126 and PD98059,
prevented ERK phosphorylation, AG126 did not. In contrast, the MEK
inhibitors did not prevent IL-1
and IL-18 release, but AG126 did.
Therefore, the results indicate that the activation of
mitogen-activated protein kinase pathway is not essential for ATP-induced IL-1
release from LPS-primed monocytes.
The Mechanism of the AG126 Effect--
It is possible that the
tyrosine kinase activity inhibited by AG126 regulates caspase-1
activation. We therefore asked if the inhibition of ATP-stimulated
release of cytokines by AG126 is mediated by the inactivation of ICE.
To accomplish this we examined the ATP-induced processing and release
of IL-1
and IL-18 in the presence of kinase inhibitors. Western blot
analysis of the cell supernatants for IL-1
(Fig.
10A) and IL-18 (Fig.
10B) confirmed the ELISA (Fig. 9) results. As it did for
IL-1
, AG126 inhibited the processing and release of IL-18. The MEK
inhibitors, herbimycin A and the nitric-oxide synthase inhibitor LNAME,
did not affect ATP-induced processing and release of the cytokines.

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Fig. 10.
AG126 inhibits caspase-1 activation and
processing and release of IL-1 and IL-18.
LPS-primed monocytes were treated with various inhibitors as described
for Fig. 9, and supernatants were subjected to Western blot analysis
for mature IL-1 (A) and for IL-18 (B), and p20
caspase-1 (C). The position of both pro- and mature proteins
are shown for IL-18. DMSO, Me2SO.
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Finally, we examined the effect of AG126 on caspase-1 activation by
Western blot analysis. Interestingly, AG126 also inhibited the ICE
activation, as the 20-kDa active form of ICE was not detected; whereas,
herbimycin A, MEK, and nitric-oxide synthase inhibitors had no effect
on ATP-induced activation of ICE (Fig. 10C). Thus the
inhibition of IL-1
release by AG126 correlates with the inhibition of ATP-induced ICE activation, and the processing of ICE appears to be
regulated by AG126-sensitive protein-tyrosine kinases.
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DISCUSSION |
The processing of precursor IL-1
and IL-18 and their secretion
into the exterior are key regulatory events in inflammation, yet remain
poorly understood. Both proIL-1
and proIL-18 are biologically inactive proteins that require cleavage by caspase-1 for biological activation (8, 9, 32). Both IL-1
and IL-18 share significant structural homology (4, 5), and both lack a leader sequence required
for their secretion. Furthermore, although the release of mature
IL-1
and IL-18 appears to be linked to the processing of precursor
forms by caspase-1, functional ICE has not been detected in monocytes,
macrophages, or monocytes cell line THP-1 even when the processing and
release of IL-1
was induced by LPS (13, 33).
Previous studies provide strong evidence that extracellular ATP
enhances both the magnitude and the velocity of the posttranslational processing and release of proIL-1
from peritoneal murine macrophages and human and microglial cells stimulated with LPS and monocytes (15,
18, 27, 34, 35). This ATP-triggered release of IL-1
occurs via
activation of the P2X7 receptor (15, 29). The telescoping
of the release and processing events provided by ATP allow a more
detailed dissection of the events that regulate the processing and
release of IL-1
and IL-18.
In this context, we utilized this unique activity of ATP to compare
IL-18 and IL-1
processing and release. We provide evidence that
there are marked similarities between IL-1
and IL-18 in this regard,
and we used the constitutive presence of proIL-18 to evaluate the role
of priming in the release event. First of all, we show that, although
IL-18 is present in fresh monocytes, the addition of ATP alone is
insufficient to induce IL-18 release. Second, utilizing assays specific
for the mature and precursor forms of these cytokines, we demonstrate
that the ICE inhibitor, YVAD-cmk, inhibits the ATP-induced maturation
of both IL-1
and IL-18 but not their release. Third, we show that
exogenous ATP rapidly activates caspase-1 in both primed and unprimed
monocytes. Furthermore, this ATP effect is mediated through
constitutively present and functional P2X7 receptors.
Finally, we show that the ATP-induced activation is, at least in part,
dependent upon protein-tyrosine kinase activity, since it is inhibited
by the PTK inhibitor AG126 but not by nitric-oxide synthase inhibition
or by mitogen-activated protein kinase, PKC, or PKA inhibitors. Thus,
we demonstrate that both IL-1
and IL-18 are released in a similar
fashion and that LPS priming is necessary for this activation for IL-18
and by implication for IL-1
as well. Furthermore, we provide the
first report showing evidence that caspase-1 activation is regulated by
a protein-tyrosine kinase activity.
The requirement for LPS priming for the ATP induction of IL-18 was
unexpected and is particularly noteworthy. Since LPS priming was
necessary for the generation of intracellular IL-1
, we expected that
the ATP effect was simply a direct trigger of the ICE activation and
protein release pathway. However, the findings with IL-18 provide the
first evidence to suggest that ATP alone is not able to trigger either
processing or release. This LPS "priming" event was not due to an
up-regulation of P2X7 receptors, since our flow cytometry
and immunoblot analyses showed constitutive presence of
P2X7 receptors with only minimal induction by LPS (Fig.
6).
The role of LPS priming in caspase-1 activation is also worthy of
comment. As shown in Fig. 5B, ATP stimulation alone
activates caspase-1 without inducing processing of constitutive IL-18
or inducing its export from the cell (Fig. 4B, lanes
4 and 13). However, ATP stimulation after LPS priming
induces both caspase-1 activation (Fig. 5B, fourth
lane) and IL-18 processing and release (Fig. 4B,
lanes 3 and 12). These observations suggest that
the ATP effect on the release pathway is distinct from its effect on
ICE activation.
In confirmation of prior studies, we found it difficult to document the
presence of mature IL-1
or mature IL-18 inside LPS-primed, ATP-stimulated monocytes (Fig. 4, A and
B). These data suggest that the processing and release of
IL-1
and IL-18 are rapid and probably concurrent events. However,
these concurrent events are likely independent of each other, since the
caspase-1 inhibitor, YVAD-cmk, did not prevent the release of the
larger precursor forms. Nevertheless, these findings do not exclude the
possibility that caspase-1 may have a role in release that is separate
from its function as a protease. For example, in ICE knockout animals, there is a deficit in release of both the mature and precursor forms of
IL-1
(26). This raises the possibility that either caspase-1 itself
or caspase-1-affiliated molecules make up part of the protein machinery
involved in IL-1
and IL-18 release. Since caspase-1 contains a
classic caspase recruitment domain (CARD), one can speculate that
CARD-interacting molecules may make up part of this release machinery
(37). The protein-protein interactions induced by similar recruitment
domains are known to be important in the function of apoptotic caspases
(38).
Since it has been reported that freshly isolated human monocytes do not
express P2X7 receptors, we were intrigued by the
responsiveness of fresh monocytes to ATP in our experiments (20). Flow
cytometric and Western blot analysis showed that indeed monocytes
constitutively express P2X7 receptors and the addition of
LPS only marginally induced P2X7 receptor expression. The
role of the P2X7 was confirmed by several experiments.
First of all, oxidized ATP, a selective inhibitor of P2X7
receptor, completely and irreversibly blocked the ATP-mediated release
of IL-1
in LPS-primed monocytes. In addition, the effect was also
significantly inhibited by a monoclonal antibody directed at the
P2X7 receptor but not by an isotype control. Finally, the
presence of P2X7 receptors was shown by both fluorescence flow cytometry and by immunoblot analysis. Thus, monocytes are extremely responsive to ATP-induced IL-1
activation via
P2X7 receptors.
Signaling pathways regulate many cellular processes. We therefore
hypothesized that activation of protein kinases may play a crucial role
in release of IL-1
and IL-18. Using classic protein kinase
inhibitors, we screened for kinases that might control release-related
events. We were intrigued by the potent inhibition provided by the
tyrphostin AG126. AG126 is a protein-tyrosine kinase inhibitor that has
been reported to be protective of endotoxin-induced shock in a mouse
model (31). This is particularly intriguing from the perspective that
the AG126 effect mimics that seen in ICE knockout animals. AG126 blocks
both ICE processing and IL-1
and IL-18 release. This inhibition
resembles the defect seen in macrophages from ICE-deficient animals
(26). Although IL-18 and IL-1
knockout animals are not protected
from septic shock, ICE knockout animals are protected (26). Thus,
events centered at the caspase-1 molecule may be critical. In this
context, it is important to note that inhibition of ICE activity by
YVAD-cmk does not prevent release, whereas genetic deletion of
caspase-1 or treatment with AG126 does. One can hypothesize that there
are release-related molecules that are linked to caspase-1. Thus, it
appears that an aspect of caspase-1 independent of its proteolytic function is critical to release. Since AG126 inhibits both caspase-1 functions, the identification of this AG126-sensitive tyrosine kinase
will likely represent a crucial activation step in IL-1
/IL-18 processing and release, which appears to be critical to the sepsis response pathway.
In an effort to put the present findings in context, it may be helpful
to review the relationship of our results to the known signaling
pathways for ATP and LPS. For example, ATP activates phospholipase D in
THP-1 cells, p42/p44 ERK phosphorylation in astroglial cells, and
NF
B activation in microglial cells (39-41). LPS is known to
induce activation of PTK-, PKC-, PKA-, and ceramide-activated protein
kinase signaling pathways (42). LPS also induces activation of src
family of tyrosine kinases Hck, Fgr, and Lyn. However, these members of
src family of tyrosine kinases do not appear to be the targets of AG126
because the production and secretion of IL-1
, IL-6, and tumor
necrosis factor-
in mice lacking Hck, Fgr, and Lyn is normal (36).
In addition, p42/p44 ERK is not required for the ATP-activated release
of IL-1
and IL-18, since PD98059 and UO126 did not prevent
processing and release but did block ERK phosphorylation. Thus the
protein-tyrosine kinase inhibited by AG126 remains uncharacterized.
Taken together these findings suggest that the regulation of the
processing and release of IL-1
and IL-18 is similar and critical to
the outcome in endotoxin responses. More importantly, these findings
suggest that that this regulatory event is linked to caspase-1
activation. Thus, the molecular events that control the processing and
release of IL-1
and IL-18 are likely to be similar and to be
critical to regulation of inflammatory responses.