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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 2 (January 15), 1998:
pp. 577-584
Lipopolysaccharide Activates Caspase-1 (Interleukin-1-Converting
Enzyme) in Cultured Monocytic and Endothelial Cells
By
Ralf R. Schumann,
Claus Belka,
Dirk Reuter,
Norbert Lamping,
Carsten J. Kirschning,
Joerg R. Weber, and
Dagmar Pfeil
From the Molecular Sepsis Research Laboratory,
Max-Delbrück-Center for Molecular Medicine, the Institute of
Microbiology and Hygiene, and the Department of Neurology, University
Hospital Charité, Humboldt-University, Berlin, Germany; and the
Department of Radiation Therapy, University of Tübingen,
Tübingen, Germany.
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ABSTRACT |
Interleukin-1 (IL-1) is a pleiotropic proinflammatory
cytokine. Mechanisms leading to its secretion include not only release of newly synthesized protein, but also cleavage of a preformed immature
precursor protein into an active secretory form by the intracellular
protease caspase-1 (formerly termed IL-1-converting enzyme [ICE]).
Caspase-1 belongs to a rapidly growing family of cysteine proteases
with substrate specificity for aspartate involved in cellular
apoptosis. We have used an assay determining the caspase-1 activity
based on cleavage of a fluorogenic peptide substrate to elucidate its
role in lipopolysaccharide (LPS)-induced secretion of IL-1. We show
that LPS induces moderate caspase-1 activity in the monocytic cell line
THP-1, in freshly isolated peripheral blood monocytes, and in human
umbilical vein endothelial cells (HUVECs) in a time- and dose-dependent
fashion. Caspase-1 activation by LPS was associated with cleavage of
the IL-1 precursor protein that was followed by release of the
mature IL-1 protein in monocytic cells. In contrast, subsequent
release of IL-1 by HUVECs was not significant. LPS-induced caspase-1
activation appeared not to result from modulation of caspase-1
transcript accumulation and inhibition of caspase-1 activity was
accomplished by two specific inhibitors, YVAD-CHO and YVAD-CMK, capable
of alleviating the release of mature IL-1. Taken together, these
results show that LPS moderately activates caspase-1 and
that caspase-1 activation contributes to LPS induction of IL-1
secretion.
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INTRODUCTION |
THE INTERLEUKIN-1 (IL-1) family of
cytokines consists of structurally and functionally related molecules
with pleiotropic activities involved in immune and inflammatory
responses.1 IL-1, one member of this family, has been
implicated in a wide range of physiologic and pathologic processes,
including mitogenic T-cell stimulation, wound healing, cellular
adhesion, cytokine and eicosanoid production, inflammation, and
sepsis.2-4 Other members of this family include IL-1 and
a naturally occuring antagonist, referred to as IL-1 receptor
antagonist (IL-1RA), which share sequence homologies and the usage of
similar receptors.3 The IL-1 family members are produced by
various cell types. Monocytes and macrophage are the major source of
IL-1. IL-1 and IL-1 target the same cellular receptor, which
is referred to as the type I IL-1 receptor (IL-1RI). Engagement of
IL-1RI leads to signal transduction, whereas the IL-1 receptor type II
is thought to act as a decoy receptor.5 IL-1 is
intracellularly cleaved and exported in large quantities upon
stimulation of its producer cell, whereas IL-1 is synthesized in
much lower quantities and does not appear to be actively
secreted.6,7
IL-1 is synthesized as an inactive 31- to 33-kD precursor protein
(pIL-1) that is cleaved by a protease to generate the mature
secretory protein (mIL-1).8,9 A cysteine protease responsible for cleavage of IL-1 has been recently identified and
termed IL-1-converting enzyme (ICE).10,11 Several
enzymes sharing sequence homology and the ability to cleave proteins at an Asparagine-site have been identified during the past
years and were recently named caspases.12
Besides ICE, which was assigned to the name caspase-1, nine other
caspases are known, with several ones being involved in the regulation
of programmed cell death (apoptosis). However, recently, it was shown
that caspase-1 does not play a role in Fas-mediated apoptosis and
intracellular IL-1 was found to be an inhibitor of apoptosis,
leaving the role of caspase-1 during apoptosis
unclear.13-15 Caspase-1 cleaves the 31-kD precursor protein
(pIL-1) at Asp116-Ala117, whereby it creates the 17.5-kD mature
biologically active cytokine.16 It also cleaves the
substrate at Asp27-Gly28 to yield products of 28 kD whose physiologic
relevance is, however, presently not understood.11,16-18 Although caspase-1 is unable to cleave IL-1,19 it was,
however, recently found to be capable of cleaving an inactive precursor of interferon- -inducing factor (IGIF; or IL-18) with high
efficiency.20 Caspase-1 thus is also involved in regulation
of interferon- production and subsequent activation of T cells. It
furthermore was found recently that in vascular smooth muscle cells an
inhibitor of caspase-1 is present that suppresses the release of mature IL-1 in the presence of ICE.21 The most
potent reversible inhibitor of caspase-1 activity is the specific
tetrapeptide aldehyde acyl-Tyr-Val-Ala-Asp-CHO or
YVAD-CHO,22 which, by forming a thiohemiacetal with the
active cysteine site, prevents release of mIL-1 by
lipopolysaccharide (LPS)-activated blood monocytes.23
YVAD-CHO selectively binds to the substrate-recognition sequence of
caspase-1. Another potent and selective irreversible caspase-1
inhibitor is the -substituted ketone chloromethylketone (YVAD-CMK)
with the general structure, acyl-Tyr-Val-Ala-Asp-chloromethylketone.
Inhibition of cysteine protease activity of caspase-1 is achieved by
expulsion of the carboxylate group to form a thiomethylketone with the
active site Cys 285.24
Unlike IL-1, caspase-1 is constitutively expressed by its producer
cells.25 The major form of caspase-1 represents a 45-kD precursor protein (p45) found in the cytoplasm that completely lacks
cleavage activity. Active caspase-1 has been purified from THP-1
monocytic cells and has been shown to consist of an equimolar ratio of
10- and 20-kD proteins, termed p10 and p20.26 LPS
stimulation of human monocytes or THP-1 cells fails to change the
amount of p45 or its activity and does not induce appearance of
detectable p20 caspase-1. cDNA cloning showed that both forms (p10 and
p20) were found within the p45 precursor protein containing four
cleavage sites, leading to formation of the protein subunits that are
flanked by Asp-X bonds, so that it is conceivable to assume that the
proenzyme is activated autocatalytically.16 Cleavage and
activation of the native p45 caspase-1 precursor has been recently
characterized by the use of specific inhibitors and antibodies
recognizing various regions of caspase-1.27 Low-level
expression and instability led to problems not only in isolation and
purification of caspase-1, but also in estimating its activity in cells
or cell lysates.25
One of the strongest inducers of IL-1 secretion is LPS (endotoxin), a
component of cell walls of Gram-negative bacteria.28 LPS,
bound by the LPS binding serum protein (LBP), is transported to its
cellular receptor, the CD14 molecule, where it induces its specific
cellular responses.29,30 Responses of
CD14 cells, eg, endothelial cells, to LPS
stimulation require the presence of soluble CD14
(sCD14).31,32 To examine LPS-induced caspase-1 activation
in different cell types, we have used a caspase-1 bioassay employing a
fluorogenic substrate that contains a specific peptide sequence of
IL-1, acyl-Tyr-Val-Ala-Asp-AMC.16 Cleavage activity of
caspase-1 is shown by increased concentrations of the fluorogenic
moiety of the substrate, aminomethylcoumarin (AMC), which can be
visualized by fluorimetry. Using this assay, caspase-1 activity can be
precisely monitored. We show that LPS induces biologically active
caspase-1 in monocytic and endothelial cells, which spurs the
generation and release of mature IL-1.
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MATERIALS AND METHODS |
Culture and cell treatment.
Human umbilical vein endothelial cells (HUVECs) were obtained
and characterized as described before.33,34 Cells were
maintained and stimulated by LPS as indicated. Monocytes were obtained
from healthy voluntary donors, separated by density gradient
centrifugation, and washed with RPMI 1640 medium. Separated cells were
resuspended in RPMI1640 containing 1% penicillin/streptomycin, 1%
glutamine, 1% sodium pyruvate, and 10% human AB serum. Aliquots of 5 mL were incubated for 2 hours at 37°C in a humidified atmosphere of
5% CO2 in air. Nonadherent cells were discarded and
adherent cells were washed with RPMI 1640 before stimulation with LPS
(100 ng/mL) in RPMI. The human monocytic leukemia cell line THP-1 was
obtained from the American Type Culture Collection (Rockville, MD).
Cells were grown in RPMI 1640 medium containing 1%
penicillin/streptomycin, 1% glutamine, 1% sodium pyruvate, and 10%
fetal calf serum. To induce differentiation associated with increased
surface expression of CD14, cells were exposed to 80 nmol/L
DH-VD3 for 3 days at 37°C.35 Cells were
stimulated with different concentrations of LPS using a medium
containing 10% AB serum. Viability and function of cells were
optimized by maintenance of cells in the log phase of growth and
monitoring for greater than 95% viability as indicated by trypan blue
exclusion.
Inhibition of enzyme activities.
Caspase-1 activity was inhibited in vivo and in vitro by addition of
the reversible inhibitor acyl-Tyr-Val-Ala-Asp-CHO or the irreversible
inhibitor acyl-Tyr-Val-Ala-Asp-chloromethylketone at concentrations of
0.2 to 20 µmol/L. The inhibitors were applied 2 hours before LPS
stimulation (in vivo) and 5 minutes before caspase-1 substrate addition
(in vitro), respectively. Viability of cells was monitored by trypan
blue exclusion and calculated by the percentage of trypan blue-positive
cells divided by protein content.
Lysis of cells.
After washing with ice-cold phosphate-buffered saline (PBS) nonadherent
cells were resuspended at 106 cells/100 µL of ice-cold
lysis buffer, and adherent cells were scraped off the plate into 100 µL lysis buffer/10 cm2 culture plate surface. Lysis
buffer consisted of 20 mmol/L Tris-acetate, pH 7.5, 1% Triton X-100,
0.1 mmol/L EDTA, 1 mmol/L EGTA, 1 mmol/L Na3VO4, 10 mmol/L
Na--glycerophosphate, 50 mmol/L NaF, 5 mmol/L Na-pyrophosphate, 1 mmol/L benzamidine, 270 mmol/L sucrose, 1 mmol/L
phenylmethyl sulfonyl fluoride, and 20 µmol/L leupeptin. Lysates were
placed on ice for 20 minutes, followed by centrifugation at
10,000g for 15 minutes at 4°C. The postmitochondrial
supernatant fraction was removed, frozen in liquid nitrogen, and stored
in aliquots at  80°C. Protein concentration of samples was
determined by the Bradford method using bovine serum albumin (BSA) as
standard.36
Electrophoresis and immunoblotting.
Protein samples of 50 µg were prepared for electrophoresis as
described.34 Samples were separated on 12% or 15%,
respectively, sodium dodecyl sulfate (SDS)-polyacrylamide gels using a
buffer system as previously described.37 After
electrophoresis, the gel was soaked in transfer buffer containing 48 mmol/L Tris, 39 mmol/L glycine, and 0.04% SDS for several minutes and
the proteins were transferred to Hybond-C extra membranes by semidry
blotting. Membranes were blocked overnight at 4°C with 5% BSA in
Tris-buffered saline (TBS) containing 0.1% Tween 20 (TBST). They were
washed intensively according to the manufacturer's instructions
(Amersham, Braunschweig, Germany), incubated with polyclonal rabbit
antihuman IL-1 antiserum, and incubated for 2 hours at room
temperature. After washing, membranes were incubated with horseradish
peroxidase-linked protein A (protein A-POD) in TBST (1:10,000)
containing 1% BSA for 90 minutes at room temperature. After a final
washing, blots were developed using the ECL system (Amersham) and
immunoreactive proteins were visualized on film.
Quantitation of mIL-1 levels by enzyme immunoassay
(EIA) and immunoprecipitation of IL-1.
The TiterZyme IL-1 EIA kit (Biermann GmbH Diagnostica, Bad Nauheim,
Germany) was used for quantitative determination of human IL-1
levels in cell culture supernatants. This assay was performed according
to manufacturer's guidelines. A total of 1.5 mL supernatant also was
incubated with 15 µg of a polyclonal anti-IL-1 antibody (Genzyme,
Cambridge, MA), followed by protein A/G plus agarose (Santa Cruz
Biotechnology, Inc, Santa Cruz, CA) addition and centrifugation according to the manufacturer's instructions. The pellet was taken up
in sample buffer and run on a 15% SDS-polyacrylamide gel
electrophoresis (SDS-PAGE), followed by a IL-1 ECL Western blot as
described above.
Measurement of caspase-1 activity.
The assay measuring caspase-1 activity in cell lysates is based on
published protocols.16,26,38 Test volumes of 100 µL consisted of 100 µg protein in a final buffer of 50 mmol/L
Tris-acetate, pH 7.4, containing 1 mmol/L dithiothreitol, 0.5 mmol/L
EDTA, and 20% glycerol. Ten microliters of a stock solution of the
acetylated and AMC-conjugated peptide substrate,
acetyl-Tyr-Val-Ala-Asp-7-amino-4-methyl-coumarin (1 µg substrate/µL
dimethyl sulfoxide), was added to the test tubes. The final
concentration was 150 µmol/L. Ten minutes after incubation at
37°C, the reaction was terminated by quick-freezing in liquid
nitrogen. Triplets of samples and the substrate control containing no
protein were thawed immediately before recording fluorescence
intensity. The reaction product AMC was detected at  ex = 350 nm and em = 435 nm. The absorbance was correlated to
caspase-1 concentration by using a standard curve of AMC. Specific activity was read as units per milligram of protein. One unit was
defined as an activity that releases 1 pmol AMC per minute.
Statistical analysis of the results.
Results shown are representative of at least three independent
experiments. Statistical analysis was performed at a level of
probability of .01 < P < .05 (caspase-1 measurements) or
P < .02 (measurements of IL-1 release), respectively, by
using the Student's t-test.
Reagents.
L-glutamine, penicillin, streptomycin, and PBS Dulbecco's solution
were obtained from GIBCO BRL, Life Technologies GmbH (Eggenstein, Germany). RPMI1640 medium, sodium pyruvate, and EDTA were purchased from Biochrom KG (Berlin, Germany). The anti-CD14 monoclonal antibody MY4 was from Coulter Electronics GmbH (Krefeld, Germany). Horseradish peroxidase-linked protein A, Hybond-C extra membranes and Rainbow colored protein molecular weight markers were obtained from
Amersham-Buchler (Braunschweig, Germany). Salmonella
minnesota Re595 LPS, sodium fluoride, sodium-orthovanadate, fetal
calf serum, and human AB serum were from Sigma-Aldrich (Deisenhofen,
Germany). Acyl-Tyr-Val-Ala-Asp-AMC (YVAD-AMC), acyl-Tyr-Val-Ala-Asp-CHO
(YVAD-CHO), and acyl-Tyr-Val-Ala-Asp-chloromethylketone (YVAD-CMK) were
purchased from Bachem Biochemica GmbH (Heidelberg, Germany).
7-amino-4-methylcoumarin (AMC) was from Fluka (Neu-Ulm, Germany).
Rabbit antihuman IL-1 antibodies were from Biermann GmbH Diagnostica
(Bad Nauheim, Germany) or Genzyme Corp (Cambridge, MA).
1,25-dihydroxyvitamin D3 (DH-VD3) was obtained
from Biomol (Hamburg, Germany). All other chemicals were of analytical
grade and were purchased from Carl Roth GmbH & Co (Karlsruhe, Germany).
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RESULTS |
LPS-induced caspase-1 activation in DH-VD3-treated THP-1
cells requires the presence of serum.
In the first set of experiments aimed at assaying for caspase-1
activity, the fluorescence intensity of the caspase-1 cleavage product
AMC was monitored to obtain a linear standard curve (not shown). The
monocytic THP-1 cell line was prestimulated with DH-VD3 to
induce differentiation towards a macrophage-like phenotype and to
induce surface expression of the cellular LPS receptor CD14. Cells were
then stimulated with 100 ng/mL LPS in the absence or presence of 10%
human AB-serum. Caspase-1 activity was not detectable in lysates of
uninduced THP-1 cells or in lysates of THP-1 cells that were cultured
in the absence of serum; however, it was readily detectable after
incubation of DH-VD3-pretreated cells in a medium
containing serum and 100 ng/mL LPS for 18 hours (not shown)
LPS-induced caspase-1 activity is concentration- and time-dependent
and is associated with intracellular accumulation and release of mature
IL-1.
Induction of caspase-1 activity in DH-VD3-pretreated THP-1
cells was time-dependent and dependent on the concentrations of LPS
used. As shown in Fig 1, the addition of
LPS to THP-1 cell cultures increases caspase-1 activity, with 100 ng
LPS/mL being the optimum stimulatory concentration. Further increase of
the LPS concentration was not associated with better inducibility of
caspase-1. Time- and dose-dependency of caspase-1 induction also
correlated with levels of IL-1 detectable in supernatants of THP-1
cells as assessed by enzyme-linked immunosorbent assay (ELISA; Fig 1B).
To examine whether LPS-induced activation of caspase-1 was also
associated with increases of intracellular levels of mature IL-1,
Western blot analysis was performed using an anti-IL-1 antibody and
cell lysates of stimulated and nonstimulated THP-1 cells. As shown in
Fig 2, the addition of LPS led to an increase of both
cleavage products of caspase-1, the 18-kD mature IL-1 protein, and
the 28 kD fragment. An increase of the 31/33-kD precursor protein was
also observed, indicating that LPS was inducing both synthesis of the
IL-1 precursor protein and activation of caspase-1, leading to
enhanced cleavage of the precursor.
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| Fig 1.
Dose- and time-dependent activation of caspase-1 and
induction of IL-1 secretion by LPS in the monocytic cell line THP-1. DH-VD3-pretreated THP-1 cells were stimulated with
increasing concentrations of LPS (S minnesota Re595) as
indicated. (A) Cells were lysed at the time points indicated and
caspase-1 activity was assessed as described in the Materials and
Methods. (B) In parallel, cell-culture supernatants were collected and
assayed for IL-1 by ELISA. Shown are mean values of three
independent experiments ± SD. (×) Without LPS; ( ) 1 ng/mL LPS;
( ) 10 ng/mL LPS; ( ) 100 ng/mL LPS; ( ) 1,000 ng/mL LPS.
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| Fig 2.
Western blot analysis probing IL-1 in cell lysates of
unstimulated and LPS-stimulated THP-1 cells. An amount of 50 µg/lane of cell lysates obtained from DH-VD3-pretreated THP-1
cells was blotted and incubated with an anti-IL-1 antibody as
described in the Materials and Methods. The left panel represents
lysates of unstimulated cells, whereas the right panel depicts lysates of cells stimulated with 100 ng/mL of S minnesota Re 595 LPS. Marked are sizes of the IL-1 precursor protein (33 kD) and the caspase-1 IL-1 cleavage products of 28 kD and 18 kD. The 18-kD fragment represents the mature secretory IL-1 (mIL-1).
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LPS induces moderate caspase-1 activity in freshly isolated
peripheral blood monocytes and HUVECs, correlating with release of
mature IL-1 in monocytes only.
To assess whether LPS induces caspase-1 activity also in freshly
isolated monocytes or in LPS-responsive, yet CD14
cells such as endothelial cells, experiments were performed with freshly isolated peripheral blood-derived human monocytes and HUVECs.
The results, which are shown in Fig 3, indicate that, in
monocytes, an approximately twofold, significant increase of caspase-1
activity can be achieved by stimulating cells with LPS and that this
increase is paralleled by a twofold enhanced IL-1 secretion (Fig
3A). In HUVECs, constitutive levels of IL-1 release could not be
significantly enhanced by LPS. However, caspase-1 activity could be
moderately increased by addition of LPS, which also was statistically
significant (Fig 3B). To determine whether mature IL-1 or proIL-1
was released into the supernatant of monocytes and HUVECs upon
stimulation by LPS, we performed an immunoprecipitation experiment,
followed by Western blotting. In both cell types, no precursor IL-1
molecule, which would migrate at 31 kD, could be detected
(Fig 4). However, mature
IL-1 at 18 kD gave rise to a signal, and the previous result of an
inducibility in monocytes but not in HUVECs was confirmed in this
assay.
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| Fig 3.
LPS induces ( ) caspase-1 activity and () mIL-1
release in freshly isolated peripheral blood-derived human monocytes
and HUVECs. Cells were isolated and cultured in vitro as described in
the Materials and Methods and stimulated with 100 ng/mL of LPS (S
minnesota Re 595). (A) Freshly isolated human monocytes exhibited a
constitutive level of IL-1 release of approximately 950 pg/mL and a
constitutive caspase-1 activity of 8 U/mg that both remained constant
over time when cells were not stimulated (not shown). Monocytes were
assayed for caspase-1 activity of lysates and mIL-1 release
into the culture supernatant at the time points indicated. (B) HUVECs
were assayed for caspase-1 activity of lysates and mIL-1 release
into the culture supernatant after 24 hours in the absence and presence
of LPS. Constitutive levels of IL-1 release and caspase-1 activity did
not change over time in the absence of LPS (not shown). Shown are the
mean values of three independent experiments ± SD. Asterisks indicate
a statistically significant difference compared with controls ([A]
were considered significant with .01 < P < .02 and [B]
with P < .05, respectively).
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| Fig 4.
Immunoprecipitation followed by Western blotting of
supernatants of HUVECs and THP-1 cells. HUVECs and THP-1 cells were
stimulated with 0.1 µg/mL LPS for 4 hours and 1.5 mL of the
supernatant was immunoprecipitated with a polyclonal anti-IL-1
antiserum, followed by Western blotting as described in the Materials
and Methods. No signal in any lane could be observed at 31 kD, the size
of the IL-1 precursor molecule, whereas at 18 kD a signal can be seen in HUVECs and stimulated THP-1 cells. At 23 kD, the lower band of
Ig can be observed. The position of the Ig heavy chain is 43 kD.
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Specific caspase-1 inhibitors block LPS-induced activation of
caspase-1 and IL-1 release in THP-1 cells.
To determine whether LPS effects on the caspase-1 activation are
specific, two peptide inhibitors were instrumental, a reversible inhibitor, YVAD-CHO, and an irreversible one, YVAD-CMK. To exclude cytotoxic effects, cell viability was assessed by trypan blue exclusion
and no cytotoxicity of both inhibitors could be observed (Table 1). YVAD-CHO and YVAD-CMK were added in vitro to
lysates of LPS-induced and control cells, respectively, and caspase-1 activity was assessed (Fig 5). Caspase-1 activity was
found to be decreased in lysates of LPS-induced and control cells by
both inhibitors in a concentration-dependent fashion. At concentrations of 0.2 µmol/L, LPS-induced increase in caspase-1 activity was blocked
significantly by both inhibitors (.01 < P < .02 for
YVAD-CHO and .01 < P < .05 for YVAD-CMK,
respectively). This condition reflects a molar ratio of 1:750
(inhibitor to substrate). Additionally, caspase-1 inhibitors were added
to the culture medium of cells, and caspase-1 activity as well as
IL-1 release induced by LPS was measured. Figure 6
shows that treatment of THP-1 cells with caspase-1 inhibitors in vivo
alleviates both LPS-induced caspase-1 activity and IL-1 release
significantly (P < .01). To exclude that caspase-1 inhibitors
affected IL-1 precursor synthesis, Western blot analyses were
performed in the absence or presence of YVAD-CMK. Figure
7 shows that the addition of the caspase-1 inhibitor failed to
significantly diminish levels of the IL-1 precursor protein. On the
contrary, addition of YVAD-CMK led to a slight increase in constitutive
levels of intracellular precursor protein. In the presence of LPS,
YVAD-CMK slightly reduced IL-1 precursor levels. As controls, the
transcriptional inhibitor of protein synthesis actinomycin D and the
translation inhibitor cycloheximide strongly suppressed synthesis of
the IL-1 precursor protein. In summary, it was shown that
LPS-induced caspase-1 actvity can be blocked specifically by the
inhibitors YVAD-CHO and YVAD-CMK.
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| Fig 5.
Inhibition of caspase-1 activity by a reversible
(YVAD-CHO) and an irreversible (YVAD-CMK) caspase-1 inhibitor measured
in lysates of DH-VD3-pretreated THP-1 cells.
DH-VD3-pretreated THP-1 cells were stimulated with 100 ng/mL of S minnesota Re 595 LPS as indicated. Cell lysates were
prepared and caspase-1 activity was assessed 18 hours after stimulation
as described in the Materials and Methods. The inhibitors were added in
vitro to the lysates 5 minutes before measurement of caspase-1 before
adding YVAD-AMC. (A) Results obtained with the reversible caspase-1
inhibitor YVAD-CHO. (B) Results obtained with the irreversible
caspase-1 inhibitor YVAD-CMK. Shown are mean values of three
independent experiments ± SD. Differences in (A) were significant
with .01 < P < .02 and in (B) with .01 < P < .05, respectively. () Without LPS; () with LPS.
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| Fig 6.
The inhibition of caspase-1 activity by specific
inhibitors in DH-VD3-pretreated THP-1 cells is paralleled
by a decrease in the release of mIL-1.
DH-VD3-pretreated THP-1 cells were stimulated with 100 ng/mL of S minnesota Re 595 LPS in the presence of the caspase-1 inhibitors YVAD-CHO and YVAD-CMK, respectively, as indicated. Cell lysates and supernatants were prepared 18 hours after stimulation. (A) Caspase-1 activity assessed as described in the Materials and
Methods. (B) Amount of IL-1 released as assessed by ELISA. Shown are
mean values of four independent experiments ± SD. Differences are
significantly with P < .01. () Without LPS; () with
LPS.
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| Fig 7.
Western blot analysis of pIL-1 in lysates of
DH-VD3-pretreated THP-1 cells that were treated with
various inhibitors before LPS stimulation.
DH-VD3-pretreated THP-1 cells were stimulated with 100 ng/mL of S minnesota Re 595 LPS in the absence or presence of
various inhibitors, as indicated. Total lysate (30 µg protein/lane) was fractionated by SDS-PAGE, transferred to nitrocellulose, and immunoblotted. The 33-kD IL-1 precursor protein reacts with an anti-IL-1 antibody as described in the Materials and Methods. Concentrations of the inhibitors used were 5 ng/mL actinomycin D, 5 µg/mL cycloheximide, and 20 µmol/L for YVAD-CHO and
YVAD-CMK.
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DISCUSSION |
Induction of cytokine production by LPS has attracted much attention in
the past because of the profound stimulatory effects that LPS exerts on
cells of the monocyte/macrophage and the endothelial cell lineages.
Although this stimulatory effect may have protective effects in the
physiologic host response to inflammatory challenges, if unbalanced, it
may contribute to acute and chronic pathologic states of the organism.
For instance, a dramatic induction of IL-1 occurs during
inflammation and sepsis and is caused by LPS. The regulation of this
process has been studied by several groups, including our
own.3,34,39 Secretion of IL-1 by monocytes is tightly
regulated, and the amount of IL-1 secreted by these cells is much
higher than that produced by any other cell type in the
body.39-41 Processing of the precursor of IL-1 to the
fully active and secretory cytokine is regulated by the specific
cystein protease caspase-1. However, up to now, regulation of caspase-1 activity has not been studied in greater detail because of technical difficulties in measuring caspase-1
activity.18,25,41,42
It has been shown that LPS enhances both the rate of transcription of
the IL-1 gene and accumulation of IL-1 transcripts. However,
enhanced gene expression alone cannot account for the rapid and strong
induction of IL-1 release by LPS seen in several cell types. We show
here that activation of caspase-1 is another mechanism whereby LPS can
induce IL-1 release. According to our results, LPS acts most likely
on both levels: transcripitonal induction of the IL-1 gene, followed
by an intracellular increase of the 30-kD IL-1 precursor, which is
paralleled by a moderate activation of caspase-1, leading to cleavage
and release of the mature IL-1 protein. Activation of caspase-1
results from a catalytic event, deliberating an inactive form of
caspase-1 into an active one. Reverse transcriptase-polymerase chain
reaction experiments performed by us suggest that a
transcriptional induction of the caspase-1 gene does not play a role
during LPS-induced caspase-1 activation (data not shown). Others have
recently shown that Langerhans cells and epidermal-derived dendritic
cell lines constitutively display high levels of caspase-1 mRNA that
are only moderately enhanced by LPS. Our data are also consistent with
studies showing that LPS-induced and noninduced THP-1 cells contain
comparable levels of caspase-1 protein, indicating that LPS does not
effect levels of caspase-1.25
It has been suggested that caspase-1 is largely associated with the
cell membrane and enhances directly the export of mature IL-1 after
cleavage of the precursor protein.18 This hypothesis is
consistent with our observation that mIL-1 is found only in small
quantities inside the cell, indicating that processing and secretion
are temporarily linked. Our data also show that the kinetics of
caspase-1 activation and secretion of IL-1 in monocytes, but not in
endothelial cells, are similar, further suggesting that the state of
caspase-1 activation in myeloid cells directly relates to secretion of
IL-1. In HUVECs, caspase-1 is also moderately induced; however, this
induction does not lead to significant IL-1 release.
Moreover, inhibition of caspase-1, which may have potential clinical
ramifications as a tool during IL-1-mediated processes, was shown
by us to inhibit IL-1 release. Inhibition by two specific compounds,
YVAD-CHO and YVAD-CMK, was almost as effective as the potent but toxic
inhibitor of protein synthesis cycloheximide (data not shown). It was
recently shown that caspase-1 (ICE) knock-out mice exhibit a striking
resistance towards experimental induction of septic
shock.43 This finding is supported by recent results
showing that caspase-1 not only leads to cleavage and release of
IL-1 but also of interferon--inducing factor (IGIF; IL-18).20 Thus, specific caspase-1 inhibitors that act
selectively and are less toxic may be valuable tools to suppress
caspase-1 activity and subsequent release of a number of cytokines and
thus may be useful in interfering with the onset and course of sepsis and septic shock. However, in our experimental approach, we were able
to inhibit only approximately 50% of LPS-induced caspase-1 activity,
and it has to be shown in animal experiments whether this inhibition is
sufficient to block the LPS effects in vivo. Experiments addressing
this question are currently underway in our laboratory.
Activation of caspase-1-like proteases has also been linked to the
induction of programmed cell death (apoptosis).44-46 During this process, activation of caspases ultimately leads to the cleavage of the poly-(ADP-ribose) polymerase (PARP) and thus to inhibition of
DNA repair. Recent findings indicate that FAS-mediated apoptosis is
mediated by other caspases, such as CPP32 or FLICE/Mach and not by
caspase-1.47-49 On the other hand, it was recently found that apoptosis induced by infection, ie, human immunodeficiency virus
infection of cells is strictly caspase-1-mediated.50
Several investigators furthermore showed that LPS can induce apoptosis in different cell types.51-54 Whether LPS-induced
activation of caspase-1 mediates programmed cell death of
monocytes/endothelial cells needs further analysis. Nonetheless, there
were no signs of apoptosis induction in the cells obseved over the
observation period reported here. Completely elucidating the cellular
events induced by LPS will aid in understanding inflammatory processes and may potentially lead to novel therapeutic strategies in sepsis and
shock.
| |
FOOTNOTES |
Submitted February 20, 1997;
accepted September 17, 1997.
Supported by Grant No. Schu 828/1-4 from the Deutsche
Forschungsgemeinschaft (DFG) and Grants No. 01KI9475 and 01KV9507 from the Bundesministerium für Bildung und Forschung (BMBF).
Address reprint requests to Ralf R. Schumann, MD, Institute of
Microbiology and Hygiene, University Medical Center
"Charité," Dorotheen Str 96, D-10117 Berlin, Germany.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section 1734 solely
to indicate this fact.
| |
ACKNOWLEDGMENT |
The authors thank Nicole Siegemund and Ina Krukenberg for their
excellent technical assistance.
| |
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Abstract
Introduction
Abstract