From the Department of Medicine, Section of Infectious Diseases, Wake Forest University School of Medicine, Winston-Salem, North Carolina 27157
Received for publication, January 12, 2001, and in revised form, March 3, 2001
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ABSTRACT |
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Microbial components such as bacterial endotoxin
lipopolysaccharide (LPS) can trigger highly lethal septic shock. The
cardinal features of septic leukocytes include the repressed production of inflammatory cytokines, such as interleukin-1 beta (IL-1 Sepsis occurs when inflammation induced by microbial infection
spreads throughout the intravascular space of humans and animals causing failure of multiple organ-systems (reviewed in Ref. 1). Sepsis
has a mortality rate of 40-80% and is the major cause of death in
critical care units in this country.
LPS1 plays a major role in
inducing sepsis during infection caused by Gram-negative bacteria (2).
Little improvement in the treatment of human sepsis has occurred since
the syndrome was defined (3). The lack of knowledge of the altered
innate immunity during sepsis may have contributed to the failures of
treating sepsis (3).
Blood leukocytes including monocytes, macrophages, and neutrophils are
exquisitely responsive to microbial infection and play important roles
in the induction of sepsis (4). The pathogenesis of sepsis during
infection depends on induction of an autotoxic and apparently
dysregulated inflammatory response from blood leukocytes including the
activation of a number of genes with both pro- and anti-inflammatory
effects (reviewed in 4). During the initial phase of bacterial
infection, a transient induction of proinflammatory cytokines including
IL-1 The septic leukocyte phenotype has also been well documented in
experimentally induced septic cell lines including human promonocytic THP-1 cells (10) as well as the murine RAW264.7 and HeNC2 macrophage cells (11). Upon initial LPS treatment, proinflammatory cytokines are
rapidly induced within these cells. Mimicking human blood leukocytes
from septic patients, prolonged treatment of THP-1 cells with LPS
induces an adapted state as reflected by the suppression of
proinflammatory proteins such as IL-1 Biochemical studies indicate that LPS-induced gene transcription
accounts for the elevated cytokine production in both human blood
leukocytes and model cell lines (12). Cytokine gene transcription is
repressed and no longer induced by LPS in septic blood leukocytes or
LPS-adapted cell lines
(12).2 Studies in our
laboratory as well as in others indicate that the differential
regulation of molecular signaling leads to an altered state of innate
immunity in septic leukocytes. Toll-like-receptors (TLRs) have been
shown in part to mediate LPS and other microbial-induced cytokine gene
expression (reviewed in 14). We observed that the interleukin-1
receptor-associated kinase (IRAK), which lies proximal in the LPS-TLR
signaling pathway, is inactivated and reduced in quantity within 3 h of LPS stimulation. The alteration of IRAK is sustained for at least
17 h in the THP-1 cell line model of the septic leukocyte
phenotype (15). Furthermore, a constitutive disruption of IRAK occurs
in blood leukocytes of patients with sepsis.3 It is likely
that a disruption in IRAK signaling contributes to the repressed
transcription of a set of LPS responsive genes, including
proinflammatory IL-1 In this investigation, we sought to identify the LPS signaling
pathway(s) that are not interrupted and control translation of sIL-1RA
in septic leukocytes. Using human blood from healthy donors and septic
patients as well as the model THP-1 cell line, we observed that the
PI3-kinase-dependent signaling pathway is still responsive
to LPS in the septic leukocyte and selectively mediates LPS-induced
translation of sIL-1RA but not IL-1 Selection of Septic Patients--
Informed consent was obtained
using a consent form endorsed by the Institutional Review Board of Wake
Forest University Baptist Medical Center. The following was used to
identify septic patients with five points or greater signifying certain
diagnosis (95% confidence) and four points signifying probable
diagnosis (>90% confidence). Major criteria (2 points
each): positive blood cultures (excluding Staphylococcus
epidermidis) and the failure of at least one of the following
organ systems: cardiovascular (systolic blood pressure <90 or low
systemic vascular resistance), pulmonary (on ventilator for respiratory
failure with diffuse pulmonary infiltrates), renal (creatine >2.5),
and microvasculature (lactic acidosis with pH <7.30 and lactate >3).
Minor criteria (1 point each): presence of local infection,
fever (>101 °F) or hypothermia (<97 °F), tachycardia (heart
rate >100), leukocytosis (white cell count >15,000) or leukopenia
(white cell count <4,000), disseminated intravascular coagulation
(increase in fibrin degradation products), and pressor agents
(dopamine, dobutamine, norepinephrine, phenylephrine, or large
quantities of fluids).
Whole Blood Analysis--
To study protein production by whole
blood leukocytes, 1 ml of whole blood was stimulated with 500 ng/ml
Escherichia coli O111:B4 LPS (Sigma) for 20 h at
37 °C in 5% CO2, and plasma was separated by
centrifugation for 5 min at 1500 × g and frozen at THP-1 Cell Culture and Induction of LPS-adaptation--
THP-1
cells were maintained in RPMI 1640 medium (Life Technologies, Inc.)
supplemented with 10 units/ml penicillin G, 10 µg/ml streptomycin, 2 mM L-glutamine, and 10% fetal bovine serum
(HyClone Laboratories, Logan, UT) at 37 °C and 5% CO2
in a humidified incubator as described previously. Low passage number
and log-phase cells were used for all experiments. LPS-adapted THP-1
cells were prepared by treating with LPS (1 µg/ml E. coli
LPS 0111:B4, Sigma) for 16 h. The cells were centrifuged, washed
once in serum-free RPMI medium, resuspended in normal RPMI medium at
1 × 106 cells/ml, and stimulated as described in the
figure legends. Cell viability for whole blood samples was determined
by trypan blue dye exclusion before centrifuging the cells at
5,000 × g for 4 min and isolating the supernatant. For
all assays, normal control cells were treated similarly but were not
exposed to LPS during the initial incubation period. When required,
cells were incubated with specific PI3K inhibitors 100 nM wortmannin (Calbiochem) or 20 µM LY294002
(Calbiochem) for 30 min at 37 °C prior to stimulation with LPS for
the times indicated. All of the results are typical of at least
three independent experiments.
sIL-1RA and IL-1 RNA Isolation and Reverse Transcription-Polymerase Chain Reaction
Analysis--
Normal and adapted THP-1 cells were stimulated with 1 µg/ml LPS for 0 to 24 h. Total RNA was isolated from 1 × 107 cells/condition using RNA STAT-60TM
(Teltest B, Inc., Friendswood, TX) according to the manufacturer's instructions. One microgram of total RNA/condition was analyzed by
radiolabeled reverse transcription-polymerase chain reaction analysis
for 40 cycles as previously described.
Western Blot Analysis--
Normal and adapted cells were treated
with 1 µg/ml LPS for the times indicated in the figure legends. Cells
(1 × 106 cells/ml) were centrifuged and lysed in 100 µl of Nonidet P-40 lysis buffer (100 mM Tris, pH 7.4, 100 mM NaCl, 2 mM EDTA, 1% Nonidet P-40, and 1 mM phenylmethylsulfonyl fluoride). Protein concentrations
were determined using BCA Protein Assay Reagent (Pierce). Proteins (100 µg of protein/lane) were separated by SDS-PAGE (7% acrylamide)
according to the Laemmli method along with low range molecular weight
markers (Bio-Rad Laboratories), and transferred to Hybond-enhanced
chemiluminescence (ECL) nitrocellulose (Amersham Pharmacia Biotech) at
100 mA for 16 h at room temperature. The nitrocellulose membranes
were blocked for 1 h with 5% nonfat milk in 1× Tris-buffered
saline (TBS)/0.1% Tween 20 and were then probed with AKT (Upstate
Biotechnology, Lake Placid, NY) and phosphorylated AKT serine 473 (Cell
Signaling, Beverly, MA) antibodies diluted in 5% nonfat milk in 1×
TBS/0.1% Tween 20. The membranes were washed three times in 1×
TBS/0.1% Tween 20 and incubated for 2 h with anti-sheep IgG
(Sigma) or anti-mouse IgG (Cell Signaling) conjugated to horseradish
peroxidase. The membranes were washed six times in 1× TBS/0.1% Tween
20 and AKT, or phosphorylated AKT was visualized using the
SuperSignal® West Pico Chemiluminescence Substrate (Pierce).
Methionine-labeling Experiments Assaying sIL-1RA
Synthesis--
1 × 107 normal and adapted cells were
untreated or pretreated with wortmannin (100 nM) for 30 min
and then stimulated with LPS (1 µg/ml) for 4 h. At the end of
the 4-h stimulation, each sample was radiolabeled with 300 µCi of
[35S]methionine in 2 ml of methionine-free medium (Life
Technologies, Inc.) for 30 min. Following the radiolabel pulse, the
radiolabeled cells were chased for 0 and 12 h with 2 ml of
nonradiolabeled complete medium. Cells were then pelleted at 1000 × g for 10 min, and radiolabeled supernatants were
collected. Supernatants were further cleared of cell debris by
centrifugation for 5 min at maximum speed in a microcentrifuge. 20 µg
of polyclonal anti-sIL-1RA (R&D Systems, Inc.) were added to 2 ml of
the radiolabeled supernatant and incubated at 4 °C for 16 h on
a rotator. 50 µl of a 50% slurry of prewashed protein G-agarose
beads (Bio-Rad) were then added to each sample followed by incubation
for an additional 16 h at 4 °C. The samples were spun briefly
in a microcentrifuge and washed six times in 1× PBS. Each sample was
solubilized with 1× SDS sample buffer (80 mM Tris-HCL, pH
6.8, 2% SDS, 50% glycerol, 0.05% bromphenol blue, and 0.2 M dithiothreitol), separated by SDS-PAGE, transferred to
polyvinylidene difluoride membrane (Bio-Rad), and visualized by autoradiography.
Statistical Analysis and Data Expression--
A mean
constitutive activity or fold induction was determined for each
experiment. Data are presented as the mean ± S.E. Statistics were
performed using either two-tailed paired or non-paired t tests to determine significant changes in activities. Data were analyzed using Microsoft Excel 97 Software (Microsoft, Seattle, WA).
sIL-1RA and IL-1
Human THP-1 promonocytic cells have been used as a model to study the
molecular mechanism of LPS adaptation that occur in sepsis (10). We
observed a similar pattern of differential IL-1 Efficient Translation of sIL-1RA Message Contributes to LPS-induced
sIL-1RA Protein Production in Adapted THP-1 Cells--
We have
previously shown that transcription of both IL-1
To further confirm that LPS induces efficient sIL-1RA translation in
adapted leukocytes, we conducted methionine-labeling experiments
assaying LPS-induced de novo sIL-1RA protein synthesis in
normal and adapted THP-1 cells. Normal and LPS-adapted THP-1 cells were
washed with fresh RPMI medium and subsequently incubated in RPMI medium
supplemented with [35S]methionine. Cells were stimulated
with 1 µg/ml LPS for 12 h at 37 °C. sIL-1RA protein was
subsequently immunoprecipitated and analyzed on SDS-PAGE. Newly
synthesized sIL-1RA proteins were detected by autoradiography. As shown
in Fig. 2B, LPS induced marked increase of
[35S]methionine incorporation into sIL-1RA proteins in
normal THP-1 cells. Despite low IL-1RA message level in adapted THP-1
cells, we observed that LPS induced similar
[35S]methionine incorporation into sIL-1RA protein in
adapted THP-1 cells compared with normal THP-1 cells (Fig.
2B). Taken together, our study indicated that the
translational efficiency of sIL-1RA message was enhanced in adapted
THP-1 cells.
The PI3-Kinase Pathway Contributes to the Enhanced Production of
sIL-1RA Protein in Septic Blood and LPS-adapted THP-1 Cells--
The
PI3-kinase-mediated signaling pathway has been implicated in regulating
protein translational efficiency (reviewed in 16). Because PI3-kinase
has been shown to be activated by LPS in murine leukocytes as measured
by Akt phosphorylation (17), we therefore tested whether LPS-induced
sIL-1RA protein production in human septic blood as well as LPS-adapted
THP-1 cells is mediated by the PI3-kinase pathway. Wortmannin and
LY294002 can specifically inhibit PI3-kinase activation and have been
used extensively to study the involvement of PI3-kinase pathway in
various biological processes. We therefore employed these inhibitors to
study the involvement of PI3-kinase pathway. Whole blood cells
collected from healthy and septic patients were treated with either
wortmannin or LY294002 and subsequently stimulated with LPS (500 ng/ml)
for 24 h at 37 °C. The sIL-1RA and IL-1
We further studied the effect of wortmannin on LPS-induced sIL-1RA
protein production in adapted model THP-1 cells. Adapted THP-1 cells
were washed and resuspended in LPS-free RPMI medium. Wortmannin was
then added to the medium to the final concentration of 100 nM. Adapted THP-1 cells pretreated with wortmannin were subsequently challenged with 1 µg/ml LPS for various amounts of time.
Consistent with the finding from septic blood cells, wortmannin pretreatment dramatically decreased LPS-induced sIL-1RA production in
adapted THP-1 cells (Fig. 3B). Similarly, IL-1
In parallel, we also assayed the phosphorylation status of Akt, the
downstream target of PI3-kinase. Following LPS and/or wortmannin
treatment of the adapted THP-1 cells, total protein extracts were
prepared and analyzed by SDS-PAGE. The levels of total Akt and Akt
phosphorylated at serine 473 were determined through Western blot using
antibodies against total Akt and phosphor-Akt-ser473 (Upstate
Biotechnology). As shown in Fig. 3C, we first observed that
LPS induced Akt phosphorylation in adapted THP-1 cells. As expected,
wortmannin pretreatment abolished LPS-induced Akt phosphorylation (Fig.
3C).
The PI3-kinase Pathway Mediates sIL-1RA Protein Translation without
Interfering with Its Transcription--
PI3-kinase activation has been
reported to regulate diverse biological processes including
transcription and translation (reviewed in 18). To determine whether
the PI3-kinase pathway regulates sIL-1RA production through interfering
with its transcription or translation, we measured the sIL-1RA message
level in the adapted THP-1 cells through Northern analyses. As shown in
Fig. 4A, LPS no longer induced
sIL-1RA message in adapted THP-1 cells. Further, we observed that
wortmannin pretreatment did not affect the sIL-1RA message level of the
adapted THP-1 cells. Our finding indicates that LPS-induced PI3-kinase
pathway activation does not interfere with sIL-1RA gene
transcription.
The effect of wortmannin on sIL-1RA translation in adapted THP-1 cells
was directly monitored through methionine-labeling experiments using
[35S]methionine. LPS-adapted THP-1 cells were washed with
fresh RPMI medium and incubated in RPMI medium supplemented with
[35S]methionine. Cells with or without 100 nM
wortmannin pretreatment were stimulated with 1 µg/ml LPS for 12 h at 37 °C. Total protein extracts were prepared, and the sIL-1RA
protein was immunoprecipitated using monoclonal antibody against
sIL-1RA and analyzed on SDS-PAGE. The newly synthesized sIL-1RA protein
was detected by autoradiography. As shown in Fig. 4B, we
observed that pretreatment with wortmannin abolished LPS-induced
[35S]methionine incorporation into sIL-1RA protein in
adapted THP-1 cells. Collectively, our results indicate that
LPS-induced PI3-kinase activation specifically enhances sIL-1RA
translation, without interfering with its transcription.
Two novel observations relevant to innate immunity and sepsis are
provided by this study. First, despite repression of cytokine gene
transcription due to disrupted TLR-IRAK-mediated signaling (15), a
PI3-kinase-dependent pathway responsible for efficient sIL-1RA translation was selectively retained and remained responsive to
further LPS challenge in the septic leukocytes. Second, the LPS-responsive PI3-kinase pathway selectively controlled sIL-1RA translation not IL-1), and
elevated production of anti-inflammatory cytokines, such as secretory interleukin-1 receptor antagonist (sIL-1RA). Pro- and anti-inflammatory cytokine gene transcriptions are equally repressed in
septic leukocytes due to disruption of the LPS signaling pathway at the
level of interleukin-1 receptor-associated kinase. The selective
elevation of sIL-1RA protein in septic blood is caused by efficient
translation of residual sIL-1RA message. In this study, we report that
the LPS-inducible phosphatidylinositol 3-kinase (PI3-kinase)-dependent signaling pathway contributes to the elevated translation of sIL-1RA in septic/LPS-adapted leukocytes. We also observe that this pathway is gene specific and does not affect the
production of proinflammatory IL-1
protein.
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
and tumor necrosis factor-
(TNF-
) occurs in blood
leukocytes from septic patients. This initial phase is followed by a
state of imbalance in which leukocytes have decreased production of
proinflammatory cytokines and enhanced production of anti-inflammatory
cytokines (5). In addition, ex vivo treatment with LPS does
not stimulate IL-1
production in leukocytes from septic patients
(6). However, anti-inflammatory proteins, such as sIL-1RA (7), are
continually induced upon additional challenge with LPS in septic
leukocytes. Such imbalance may lead to an adapted state of
immunosuppression, thus increasing the mortality risk from subsequent
super infection with other microorganisms. Recent reports in both
clinical patient cases and animal models support this concept (5, 8).
We refer to this adapted state as the septic leukocyte phenotype. The
septic leukocyte phenotype can develop within hours following the onset of Gram-positive and Gram-negative infections and within 3 h when LPS is experimentally administered to humans or animals (9). The septic
leukocyte phenotype appears highly reproducible and displays consistent
features that are sustained during the clinical course of septic shock
in humans and animals (6).
and TNF-
. Upon further LPS
treatment, LPS-adapted THP-1 cells exhibit continued production of
anti-inflammatory proteins such as sIL-1RA (7).
and TNF-
. Repression of transcription and
rapid degradation of proinflammatory cytokine mRNAs contribute to
decreased proinflammatory cytokine protein production in septic blood
leukocytes and LPS-adapted cell lines.2 Despite the
disrupted TLR signaling and reduced cytokine gene transcription, LPS
can still induce sIL-1RA protein translation in adapted
leukocytes.2 This indicates that the pathway controlling
sIL-1RA translation is not disrupted and is responsive to further LPS challenge.
.
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
20 °C until ELISA analysis. When required, blood samples were incubated with specific PI3K inhibitors 200 nM
wortmannin (Calbiochem) or 30 µM LY294002 (Calbiochem)
for 30 min at 37 °C prior to stimulation with LPS for the times
indicated. All of the results are typical of at least three independent experiments.
Enzyme Immunoassays--
sIL-1RA and
IL-1
levels in culture supernatants and plasma samples were assayed
in duplicate and quantified by enzyme-linked immunosorbent assay with
Quantikine Enzyme Immunoassay Kit® (R&D Systems,
Minneapolis, MN) against human IL-1 receptor antagonist and IL-1
according to the manufacturer's instructions.
RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
Proteins Are Produced Differentially in Septic
Whole Blood and THP-1 cells--
We collected whole blood from
patients suffering acute sepsis as well as from healthy donors from
Wake Forest University Baptist Medical Center. Serum levels of IL-1
and sIL-1RA were collected and then assayed by ELISA. IL-1
and
sIL-1RA protein levels in healthy normal human blood are virtually
absent (Fig. 1A). Following LPS stimulation, IL-1
protein level increases ~50-fold while sIL-1RA protein level increases about 5-fold by 24 h. In septic blood, both IL-1
and sIL-1RA proteins are constitutively present at
low levels. Unlike normal blood, LPS does not induce IL-1
protein
production in septic blood. In marked contrast to IL-1
, sIL-1RA
protein in septic blood is induced to a similar level as in normal
blood following LPS stimulation (Fig. 1A).
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Fig. 1.
sIL-1RA and IL-1
proteins are produced differentially in septic blood as well as
adapted THP-1 cells. A, whole blood
from septic patients and controls were incubated with or without LPS
for 20 h. Plasma was subsequently separated and analyzed for
IL-1
and sIL-1Ra protein by ELISA; B, normal and adapted
THP-1 cells were washed with RPMI medium and further incubated with or
without LPS for the indicated amount of time. Total concentrations of
the sIL-1RA and IL-1
proteins were assayed by ELISA as described
under "Materials and Methods." This figure represents the average
of three independent experiments.
and sIL-1RA protein
production in THP-1 cells. In unstimulated normal THP-1 cells, there is
no detectable IL-1
and only a very low level of sIL-1RA protein
(Fig. 1B). Upon stimulation with LPS, IL-1
and sIL-1RA
protein levels were increased about 50- and 5-fold, respectively,
within a 24-h range. Prolonged stimulation with LPS rendered THP-1
cells adapted/tolerant. Resembling human blood from septic patients,
adapted THP-1 cells exhibited suppressed IL-1
production and
continued sIL-1RA production following a second dose of LPS stimulation
(Fig. 1B, Ref. 7).
and sIL-1RA is
repressed in septic leukocytes as well as in LPS-adapted THP-1 cells
(12). As shown in Fig. 2A, the
message level of sIL-1RA is low and not induced by LPS in LPS-adapted
THP-1 cells. We also observed that the message as well as protein
stability of sIL-1RA was not altered following LPS-adaptation/tolerance of THP-1 cells (data not shown). The fact that sIL-1RA protein level is
significantly increased in septic/LPS-adapted leukocytes suggests that
LPS induces efficient translation of residual sIL-1RA message in
septic/LPS-adapted leukocytes.
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Fig. 2.
LPS-induced IL-1RA production is caused by an
increase in efficient translation not in transcription.
A, normal and adapted THP-1 cells were washed with LPS-free
RPMI medium, resuspended in fresh RPMI medium, and stimulated with LPS
for the indicated amount of time. Total RNAs were isolated and analyzed
by Northern blot using radiolabeled IL-1RA and
glyceraldehyde-3-phosphate dehydrogenase cDNA; B, LPS
induces similar level of sIL-1RA protein synthesis in normal and
adapted cells. Newly synthesized 35S-sIL-1RA
proteins in resting and LPS-stimulated normal and adapted THP-1 cells
are shown. THP-1 cells were incubated in RPMI medium supplemented with
300 µCi of [35S]methionine and stimulated with LPS (1 µg/ml) for 12 h. This figure represents results from two
independent experiments
protein levels were
then assayed by ELISA. As shown in Fig.
3A, pretreatment with
wortmannin or LY294002 significantly decreases LPS-induced sIL-1RA
protein production in septic human blood cells. Interestingly, we
observed no effect of either wortmannin or LY294002 on IL-1
protein
production in parallel experiments (Fig. 3A).
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Fig. 3.
Wortmannin specifically inhibits LPS-induced
sIL-RA not IL-1 protein production in septic
blood as well as adapted THP-1 cells. A, sIL-1RA
and IL-1
protein levels were determined by ELISA in sepsis blood
treated with either LPS alone, LPS and wortmannin, or LPS plus
LY294002; B, sIL-1RA and IL-1
protein levels were assayed
by ELISA in adapted THP-1 cells treated with either LPS, wortmannin or
LPS plus wortmannin; C, adapted THP-1 cells were washed,
resuspended in RPMI medium, and incubated in RPMI medium with or
without wortmannin for 1 h. LPS was subsequently added to a final
concentration of 500 ng/ml for the indicated amount of time. Protein
extracts were isolated and resolved on SDS-PAGE followed by Western
transfer. Monoclonal antibodies against unphosphorylated and
phosphorylated Akt were used to perform the Western blot.
Akt-P, phosphorylated Akt; Akt, unphosphorylated
Akt.
production was not affected by wortmannin treatment (Fig. 3B).
Experiments using LY294002 gave similar results (data not shown).
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Fig. 4.
Wortmannin inhibits IL-1RA translation not
its transcription. A, wortmannin does not affect
sIL-1RA mRNA levels in adapted THP-1 cells. Adapted THP-1 cells
were washed with LPS-free RPMI medium, resuspended in fresh RPMI
medium, and stimulated with either LPS alone or wortmannin plus LPS for
the indicated amount of time. Total RNAs were isolated and analyzed by
Northern blot using radiolabeled IL-1RA and glyceraldehyde-3-phosphate
dehydrogenase cDNA. B, wortmannin reduces sIL-1RA
protein synthesis in LPS-adapted cells. Adapted THP-1 cells were
incubated in RPMI medium supplemented with 300 µCi of
[35S]methionine and stimulated with LPS (1 µg/ml) or
wortmannin plus LPS for 12 h. This figure represents results from
two independent experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
. Our study indicates that differential
regulation of LPS-mediated signaling pathways contributes to altered
cytokine protein profiles and the septic leukocyte phenotype (Fig.
5).
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Fig. 5.
Schematic illustration of the altered
signaling pathways in septic/adapted leukocytes.
Our findings reveal a novel LPS signaling pathway that is responsive in LPS-adapted leukocytes. In normal leukocytes, LPS triggers TLR4 receptor-mediated signaling and activates a series of kinases including IRAK as well as various mitogen-activated protein kinases (11, 15). Prior exposure to LPS was reported to render leukocytes hyporesponsive to further LPS challenge, a phenomenon also known as endotoxin tolerance, which we refer to here as LPS adaptation. LPS-adapted leukocytes were shown not to respond to further LPS challenge as measured by suppression of IRAK kinase (15) and TLR4 signaling as well as all forms of mitogen-activated protein kinases (11). Disruption of these LPS signaling events may account for the repressed cytokine gene transcription that occurs in septic leukocytes or experimentally LPS-adapted cell lines. However, LPS-adapted leukocytes still express several anti-inflammatory cytokines including IL-10 and sIL-1RA (7). This indicates that endotoxin tolerance is not a total inhibition of cellular activities but rather an adaptation or reprogramming of cellular signaling events. Until now, it is not known which LPS signaling pathway still remains open and is responsible for the continued sIL-1RA production in LPS-adapted leukocytes. We show in this report that LPS induces PI3-kinase pathway activation in LPS-adapted cells (Fig. 3C). The activation of PI3-kinase is commonly measured through the phosphorylation of endogenous Akt protein, a direct downstream target of PI3-kinase (reviewed in Ref. 19). PI3-kinase pathway activation and Akt phosphorylation was observed in LPS-stimulated normal macrophages, neutrophils, as well as monocytes (17, 20, 21). However, there has been no previous biochemical study regarding the PI3-kinase activity in septic or LPS-adapted leukocytes. In this report, we are the first to report that Akt undergoes phosphorylation in LPS-adapted THP-1 cells upon further LPS treatment.
In addition, our work provides a novel link between PI3-kinase pathway activation and sIL-1RA protein translation in septic/adapted leukocytes. The phospholipid products of PI3-kinase, phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-triphosphate, can act as second messengers and activate several downstream kinases including Akt, inositol phosphate kinase, and several calcium-dependent protein kinase C forms (18). These downstream kinases can subsequently regulate multiple cellular events including gene transcription (13) as well as protein translation (17). Among the multiple targets of PI3-kinase, the activation of Akt, mTOR, and p70S6 kinases have been well documented to regulate ribosomal assembly and protein translational efficiency (16). We show here that inhibition of PI3-kinase by either wortmannin or LY294002 abrogates sIL-1RA protein production induced by LPS in septic whole blood or LPS-adapted THP-1 cells (Fig. 3, A and B). We observed that LPS-induced sIL-1RA message levels in human septic leukocytes or LPS-adapted THP-1 cells is not affected by PI3-kinase inhibitors (Fig. 4A), suggesting that the PI3-kinase pathway selectively controls sIL-1RA translation not its transcription or message stability. We also observed that rapamycin, an inhibitor of mTOR, inhibits LPS-induced sIL-1RA protein production (data not shown). In addition, pulse-chase experiments show that inhibition of PI3-kinase significantly decreases incorporation of [35S]methionine into newly synthesized sIL-1RA protein (Fig. 4B). Our findings indicate that the PI3-kinase pathway plays a critical role in directly regulating sIL-1RA translation.
Interestingly, we observe that inhibition of PI3-kinase pathway does
not interfere with other cytokine production such as IL-1 (Fig. 3,
A and B). LPS-induced IL-1
message and protein levels in both septic blood and LPS-adapted THP-1 cells are not affected by PI3-kinase inhibitors. This indicates that the
LPS-activated PI3-kinase pathway specifically regulates a certain set
of cytokines and their translation. The mechanism of such regulation
remains to be elucidated.
Taken together, we conclude that septic/LPS-adapted leukocytes can
still respond to LPS stimulation and undergo activation of PI3-kinase
pathway. LPS-induced PI3-kinase pathway activation selectively
contributes to the efficient translation of sIL-1RA not IL-1. Our
work further underscores that the septic leukocyte phenotype is a
complex adaptation of cellular signaling events, involving not only
repression of TLR-IRAK and several other signaling pathways but also
selective activation of the PI3-kinase pathway. This altered phenotype
may represent a modification of innate immunity that could either
protect the host from further injury or produce an immunocompromised
state. Further biochemical examination of the fine interplay of these
signaling events is needed for better understanding and treatment of
sepsis and other inflammatory diseases.
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ACKNOWLEDGEMENT |
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We thank Drs. Douglas Lyes and Steven Mizel for thoughtful discussions. Cell culture and media were supplied by the Tissue Culture Core Laboratory of Wake Forest University School of Medicine.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Supported by research funding from the NCI National Institutes of
Health Training Grant CA-09422.
§ Supported by research grants from the American Lung Association. To whom correspondence should be addressed: Dept. of Medicine, Section of Infectious Diseases, Wake Forest Univ. School of Medicine, Medical Center Blvd., Winston-Salem, NC 27157. Tel.: 336-716-6040; Fax: 336-716-3825; E-mail: lwli@wfubmc.edu.
¶ Supported by National Institutes of Health Grant AI09169 and MM01 RR07122-10.
Published, JBC Papers in Press, March 28, 2001, DOI 10.1074/jbc.M100316200
2 L. Mueller, L. Li, and C. E. McCall, submitted for publication.
3 L. Li and C. E. McCall, unpublished observation.
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ABBREVIATIONS |
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The abbreviations used are:
LPS, lipopolysaccharide;
IL, interleukin;
sIL-1RA, secretory
interleukin-1 receptor antagonist;
TNF-, tumor necrosis factor-
;
TLR, toll-like-receptor;
IRAK, interleukin-1 receptor-associated
kinase;
PI3, phosphatidylinositol 3-kinase;
ELISA, enzyme-linked
immunosorbent assay;
PAGE, polyacrylamide gel electrophoresis;
TBS, Tris-buffered saline.
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