From the Division of Biopharmaceutics,
Leiden/Amsterdam Center for Drug Research, University of Leiden,
Sylvius Laboratories, P. O. Box 9503, 2300 RA Leiden, The Netherlands;
the
Research Institute Neurosciences Vrije Universiteit, Faculty
of Medicine, Department of Pharmacology, Van der Boechorststraat 7, 1081 BT Amsterdam, The Netherlands; and ** Bio-Technology General Ltd.,
Kiryat Weizmann, Rehovot 76326, Israel
Received for publication, October 31, 2000, and in revised form, January 2, 2001
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ABSTRACT |
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Septic shock is the most common cause of
death in intensive care units and no effective treatment is available
at present. Lipopolysaccharide (LPS) is the primary mediator of
Gram-negative sepsis by inducing the production of macrophage-derived
cytokines. Previously, we showed that apolipoprotein E (apoE), an
established modulator of lipid metabolism, can bind LPS, thereby
redirecting LPS from macrophages to hepatocytes in vivo. We
now report that intravenously administered LPS strongly increases the
serum levels of apoE. In addition, apoE can prevent the LPS-induced
production of cytokines and subsequent death in rodents. Finally,
apoE-deficient mice show a significantly higher sensitivity toward LPS
than control wild-type mice. These findings indicate that apoE
may have a physiological role in the protection against sepsis, and
recombinant apoE may be used therapeutically to protect against
LPS-induced endotoxemia.
Sepsis is a syndrome referring to an exaggerated systemic response
to infections, which can ultimately lead to death from septic shock. In
fact, in the United States the incidence of sepsis has increased during
the last decennia (1) and sepsis has become the most common
cause of death in intensive care units, with 150,000 deaths annually
(2, 3). Many cases of sepsis are caused by Gram-negative bacteria (1).
Lipopolysaccharide (LPS),1 a
component of the outer membrane of these bacteria, is the primary cause
of Gram-negative sepsis and gives rise to the same clinical features as
are observed in patients with sepsis (3-6). Within the blood, the
lipid A-moiety of LPS binds to the LPS-binding protein (7, 8), and the
resulting complex displays a high affinity for CD14-toll like receptor
4 (Tlr4) complex on mononuclear phagocytes (9, 10). Activation of these
cells induces the release of inflammatory mediators such as tumor
necrosis factor alpha (TNF Lipoproteins are suggested to play an important role in the protection
against infection and inflammation. All lipoproteins (high density
lipoproteins (HDL), low-density lipoproteins (LDL), lipoprotein(a),
very-low-density lipoproteins (VLDL), and chylomicrons) can bind
endotoxin (27-32) and thereby reduce the toxic properties of LPS. In
particular, incubation of VLDL or chylomicrons with LPS before
administration to rodents significantly reduces the serum levels of
TNF Triglyceride-rich lipoproteins may be used therapeutically to protect
against Gram-negative sepsis or septic shock, but the need for
isolation from human lymph or blood impedes their possible application.
Within our laboratory, we have developed an emulsion model for
chylomicrons from commercially available lipids and human recombinant
apoE (35), which is selectively taken up via apoE-specific receptors on
liver parenchymal cells. In a rat model, we demonstrated that
recombinant chylomicrons can target LPS to liver parenchymal cells,
which prevents its binding to both splenic and hepatic macrophages.
Furthermore, it was shown that apoE binds LPS directly and alters its
metabolic fate, suggesting that apoE in triglyceride-rich lipoproteins
may be crucial for the lipoprotein-endotoxin interaction (36).
In the present study we examined whether apoE is increased by endotoxin
and whether its interaction with endotoxin is part of a physiological
response. In addition, we determined whether apoE protects rodents
against the detrimental effects of endotoxin.
Rats and Mice--
Nine to ten-week-old male Wistar rats of mass
260-310 g and 10-12-week old C57Bl/6 mice of mass 21-27 g and
apoE-deficient mice (crossed back on a C57Bl/6 background; Refs. 37,
38) were obtained from Broekman Institut BV, Someren, The Netherlands and were fed ad libitum with regular chow (unless otherwise stated).
Cytokine Determination--
Emulsion was prepared as described
earlier (36). Rats were given an intravenous (i.v.) injection of LPS
(10 µg/kg) derived from Salmonella minnesota R595 (Re)
(List Biological Laboratories Inc., Campbell, CA), which was
preincubated with PBS, apoE-free emulsion (20 mg of triglycerides per
kg), apoE (800 µg/kg) or apoE-enriched emulsion for 30 min at
37 °C. Blood samples were taken up to 150 min after injection from
the tail vein and allowed to clot for 30 min at room temperature. Serum
samples, obtained after centrifugation at 16,000 × g
for 5 min, were screened for their IL-1
ApoE-deficient mice (22-27 g) and control mice (22-27 g) were given
an intravenous injection of LPS (25 µg/kg body weight). At the
indicated time points (up to 180 min), blood samples were taken, and
serum was obtained as described above. In the serum samples TNF IL-1 IL-6 Bioassay--
IL-6 was detected as described by Aarden
et al. (39). In short, the IL-6-dependent B9
cells (mouse hybridoma cell line) were cultured in IMDM
containing 5% fetal calf serum, 3.02 g/liter NaHCO3, 0.1%
TNF Mortality--
Mice received an intraperitoneal injection of 20 mg of D-galactosamine. Subsequently, they were injected
i.v. with LPS (150 ng/kg), which was preincubated for 30 min at 37 C
with PBS or apoE (25 µg/kg). During the 72-h period after injection,
the survival was determined, after which no further loss of animals occurred.
ApoE ELISA and Cholesterol Determination--
Mice were injected
with PBS or LPS (100 µg/kg). At different time points after
injection, blood samples were taken from the tail vein and allowed to
clot for 30 min. Serum was obtained by centrifugation for 5 min at
16,000 × g and screened for their apoE content, using
a mouse apoE-specific ELISA as described (36), and total cholesterol
using a commercial enzymatic kit from Roche Molecular Biochemicals
(Mannheim, Germany).
Western Blot--
Sera were obtained as described above.
Proteins within the sera (1-µl aliquots) were separated by 10%
SDS-polyacrylamide gel electrophoresis under reducing conditions and
blotted onto nitrocellulose membrane in a buffer containing 25 mM Tris, 20% methanol, 192 mM glycine, and
0.02% SDS. Blots were immunolabeled with rabbit anti-mouse apoE
antibody and visualized by enhanced chemiluminescence, essentially as
described (40).
Statistical Analysis--
The n value is indicated
for each experiment. Statistical differences in cytokine and apoE
production were determined using a two-tailed Student's t
test. Statistical differences in survival curves among the groups of
mice were analyzed by log rank test. For both analyses, Graphpad
software (Prism and Instat) was used.
Effect of apoE on LPS-induced Cytokine Levels--
To determine
the effect of apoE on LPS-induced proinflammatory cytokine levels, rats
were injected with LPS in the absence or presence of apoE and/or
emulsion, and the cytokine levels were determined up to 4 h after
injection. Injection of LPS resulted in a strong induction of TNF Effect of apoE on LPS-induced Lethality--
The proinflammatory
cytokines TNF Cytokine Response in apoE-deficient Mice--
ApoE-deficient mice
and control mice were injected with a sublethal dose of LPS (25 µg/kg). The level of TNF Effect of LPS on Endogenous apoE Levels--
Next, we questioned
whether the observed effects of exogenous apoE on the detoxification of
LPS may imply a physiological role of endogenous apoE in Gram-negative
bacterial infections. It is known that LPS, which remains in the serum
after injection into rodents, is largely associated with HDL (42). ApoE
does bind to HDL when injected into rodents, and we observed that
incubation of LPS with apoE before injection, results in an ~3-fold
higher association of LPS with HDL in rats and mice (36). We
hypothesized that LPS may increase the endogenous serum levels of apoE,
leading to an increased detoxification capacity of the serum. Indeed, a
single challenge of mice with LPS (100 µg/kg) had a profound effect
on the serum level of endogenous apoE (Fig.
4A). Whereas injection of PBS
had no effect, injection of LPS resulted in a gradual increase of the
apoE concentration up to 178.8 ± 8.4% of the initial value at
12 h after injection. The apoE level returned to baseline levels
at 36 h after injection (Fig. 4A). In these fed
animals, LPS had no substantial effect on the serum levels of
triglycerides, which basically showed feeding
state-dependent fluctuations. However, a small but
significant difference in cholesterol level was observed at 12 h
after injection (p < 0.05) (Fig. 4B). To
avoid the feeding-dependent lipid fluctuations, the
experiment was repeated with animals, which were fasted, starting
12 h before injection and throughout the experiment (not shown).
In the control animals a decrease in serum apoE (80.4 ± 7.4% of
the initial value) and total cholesterol (93.0 ± 0.1% of the
initial value) was observed. In contrast, at 100 µg/kg, LPS again
largely increased the serum level of apoE (186 ± 11%;
p < 0.001), which was accompanied by a modest, but
highly significant, increase in cholesterol level (115.8 ± 4.8%;
p < 0.01). Analysis of the apoE content of the individual lipoprotein fractions that were separated by fast protein liquid chromatography (SMART System; Amersham Pharmacia Biotech AB,
Uppsala, Sweden), showed that the additional apoE was specifically recovered in the HDL fraction, which represents the predominant lipoprotein fraction in mice (not shown).
The increase in serum apoE was also visualized by immunolabeling on
blots and was in accordance with the data obtained with ELISA (Fig.
4C). The intensity of the apoE band was increased at 12 h after the injection of LPS, whereas in the controls a slight
reduction of the apoE staining was observed. The antibody reacted
solely with a single 34-kDa protein band in the serum of fasted
C57Bl/6J mice that were injected with PBS or LPS. This band could not
be detected in the serum of apoE-deficient mice, which accounts for the
specific staining of apoE.
The present work demonstrates that LPS strongly increases the
endogenous serum level of apoE in a rodent model. The mechanism by
which apoE is increased may involve either de novo protein synthesis as induced by LPS and/or cytokines or release from existing intra- and extracellular pools. It is likely that a continuous LPS
stimulus may provoke a long-term elevation of serum apoE levels. Indeed, septic patients demonstrated that, although these patients show
a severe hypocholesterolemia, both the HDL and LDL fractions were
largely enriched in apoE (43), whereas a single LPS-challenge increased
the apoE content of HDL in African green monkeys (44).
In addition, we observed that exogenous apoE decreased the LPS-induced
production of proinflammatory cytokines, probably by decreasing the
release of cytokines by macrophages. These data are in full accordance
with our earlier findings that free and emulsion-bound apoE, but not
the emulsion alone, altered the in vivo kinetics of
radioiodinated LPS and largely decreased the association of LPS with
macrophages (36). The interference of apoE with the metabolic fate of
LPS is not only accompanied by a strong inhibition of the cytokine
levels in serum, but also protects against LPS-induced death,
indicating that the reduction in cytokine levels is followed by a
reduction in mortality. The absence of apoE from the serum
(apoE-deficient mice) led to a 2-fold higher sensitivity of the mice
for treatment with LPS than control mice, because in the absence of
apoE, a 2-fold increase is TNF Until recently, apoE has been assigned a classical anti-atherogenic
role in lipid metabolism (46). Recent data, however, indicate that only
2-10% of the endogenous apoE serum level in rodents is sufficient for
the maintenance of cholesterol homeostasis (38, 47). These data imply
that serum apoE may have other functions that are unrelated to lipid
metabolism. Indeed, initial data do indicate that apoE may have
immunomodulatory functions (48-51) and that apoE may also influence
the extension of neurites in the brain (50). The present observations
strongly suggest that we have now identified a protective role of apoE
in Gram-negative sepsis.
In conclusion, we postulate that in severe Gram-negative bacterial
infection, a physiological increase in endogenous apoE forms a defense
mechanism against the development of sepsis. If, withstanding this
protection mechanism, the infection is not adequately neutralized,
administration of exogenous apoE may be of highly therapeutic
significance to overcome failure of this endogenous defense mechanism.
INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
) and interleukins (IL-1
, IL-1
, and
IL-6). These cytokines are responsible for the metabolic and
physiologic changes that ultimately lead to pathological conditions
(11-13). The importance of these cytokines in LPS-induced death arises
from observations that administration of TNF
or IL-1 to animals
provokes a similar reaction as detected after injection of LPS
(14-17). In addition, antibodies against TNF
protect monkeys
(18-20), rabbits (21), and mice (17) against LPS-induced death. Also,
blockade of IL-1 production prevents LPS-induced death of mice (22).
Current therapeutic strategies are, therefore, directed against LPS
(bactericidal/permeability-increasing protein (BPI), antibodies against
LPS (23, 24)), cytokines (soluble TNF receptor, anti-TNF antibodies
(25)), and receptors (soluble CD14, IL-1 receptor antagonist (26),
antibodies against LBP), but the initial clinical data are merely disappointing.
and protects against endotoxin-induced death (28, 29). The
protective effect is caused by a lipoprotein-mediated redirection of
LPS from Kupffer cells to parenchymal liver cells (29, 33) and a
subsequent secretion of LPS into the bile, where it is inactivated
(34). Consequently, macrophages become less activated, which results in
a reduced production of proinflammatory mediators.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
, IL-6, and TNF
content.
levels were determined.
Assay--
Rat IL-1
was detected using a rat
IL-1
-specific ELISA. 96-well plates (NUNC MAXISORP) were coated
overnight at 4 °C with sheep anti-rat IL-1
antibodies in coating
buffer (PBS containing 0.14 M NaCl, 2.7 mM KCl,
1.5 mM KH2PO4, 8.1 mM
Na2HPO4, pH 7.2-7.4). Plates were washed three
times with wash/dilution buffer (0.5 M NaCl, 2.5 mM NaH2HPO4, 7.4 mM
Na2HPO4, 0.1% Tween 20, pH 7.2). The plates
were incubated overnight at 4 °C with serum samples and recombinant
rat IL-1
(diluted in normal rat serum) as a standard. The plates
were washed and subsequently incubated with biotinylated sheep anti rat
IL-1
for 1 h at room temperature. Plates were washed again and
streptavidin/polyhorseradish peroxidase was added. After 30 min at room
temperature, plates were washed and 1,2-o-phenylenediamine dihydrochloride was added, and plates were left for 15 min at room
temperature. The reaction was stopped by addition of 1 M H2SO4, and the absorbance was measured at 495 nm.
-mercapthoethanol, penicillin/streptavidin, and 30 units/ml human
recombinant IL-6 (Central Laboratory for Blood research (CLB),
Amsterdam, The Netherlands). For detection of IL-6, B9 cells (5 × 103 cells/well) and dilutions of serum samples were added
to 96-well plates in a total volume of 200 µl/well. Human IL-6 was
used as a standard. Cells were incubated for 72 h at 37 °C, 5%
CO2. After this period [3H]thymidine (125 nCi/well) was added, and the cells were incubated for another 4 h
at 37 C, 5% CO2. Cells were harvested on a glass filter,
and [3H]radioactivity was counted using a Wallac
MicroBeta plus LCC.
Assay--
TNF
content of the serum samples was
determined using a commercially available ELISA kit for rat or mouse
TNF
(Immunosource, Zoersel-Halle, Belgium). The assay was performed
according to the manufacturer's instructions.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
,
IL-1
, and IL-6 in serum, reaching peak levels at 60, 90, and 120 min
after LPS administration, respectively (Fig.
1). Injection with PBS alone had no
effect on cytokine levels (not shown). Administration of the
apoE-enriched emulsion together with LPS largely inhibited the
LPS-induced maximum levels of TNF
, IL-1
, and IL-6 for 80%, 91%,
and 98%, respectively (p < 0.05, Fig. 1). In fact,
apoE alone inhibited the LPS-induced maximum serum levels of these
cytokines (p < 0.05) to a similar extent (68%, 99%,
and 99%) as the apoE-enriched emulsion. The effects observed with free
and emulsion-associated apoE were not significantly different,
suggesting that the apoE moiety of the recombinant chylomicrons is
responsible for the detoxification of LPS. In agreement with this
assumption, the apoE-free emulsion did not significantly reduce the
LPS-induced cytokine levels (Fig. 1).
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Fig. 1.
Effect of apoE and/or emulsion on the
LPS-induced serum levels of proinflammatory cytokines. Rats were
given an i.v. injection of LPS, which was preincubated with PBS ( ),
apoE-free emulsion (
), apoE (
), or apoE-enriched emulsion (
).
Blood samples were taken at the indicated times from the tail vein and
allowed to clot for 30 min at room temperature. Serum was screened for
its TNF
(A), IL-1
(B), and IL-6
(C) content. Values are the mean ± S.E. of three
experiments.
and IL-1 may both play an important role in the fatal
outcome of Gram-negative sepsis. Inhibition of the LPS-induced serum
levels of these cytokines by concomitant administration of apoE is,
therefore, expected to render rodents less susceptible to a lethal dose
of LPS. Mice were sensitized to the lethal action of LPS with
D-(+)-galactosamine (GalN) according to Galanos et
al. (41). We have observed in rats that preincubation of
radioiodinated LPS (10-20 µg/kg) with apoE (1.6 mg/kg) significantly
reduced the uptake of LPS by the liver (46.1 ± 5.3%
versus 69.2 ± 7.4% of the injected dose at 10 min
after injection; p < 0.001), and increased the
HDL-bound fraction of LPS in the serum (40.5 ± 3.5%
versus 12.6 ± 2.8%; p < 0.001, Ref.
36). Using the same dosage in mice, the effects of apoE on the kinetics
of LPS are essentially similar. ApoE significantly reduces the liver
uptake of LPS (54.0 ± 2.1% versus 79.2 ± 5.0% at 10 min after injection; p < 0.01), and increases
the association of LPS to HDL in the serum (33.3 ± 1.0%
versus 12.1 ± 3.0%; p < 0.01) (not
shown). The distribution of LPS over the various organs was similar in
mice and rats. The same results were obtained using GalN-pretreated
mice, indicating that GalN does not interfere with the mechanism of
redirecting LPS from (hepatic and splenic) macrophages to liver
parenchymal cells in vivo. Intravenous injection of LPS into
GalN-pretreated mice (150 ng/kg) resulted in death of 57% (4/7) of the
mice within 48 h after injection (Fig.
2). In contrast, all mice were rescued by
the concomitant administration of a low dose of apoE (25 µg/kg),
which was just sufficient to bind all of the injected LPS (36). This
significant (p = 0.022) difference in mortality
demonstrates that the interference of apoE with the metabolic fate of
LPS is not only accompanied by a strong inhibition of the cytokine
levels in serum but also protects against LPS-induced death.
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Fig. 2.
Effect of apoE on LPS-induced mortality.
Mice, sensitized with D-galactosamine hydrochloride, were
injected i.v. with LPS, which was preincubated with PBS ( ) or apoE
(
). Survival was determined during the 72-h period after injection,
after which no further loss of animals occurred (n = 7). A Kaplan-Meier survival curve is shown. Asterisk
indicates a significant difference (p = 0.022) between
the LPS-treated animals and the LPS-apoE-treated animals.
was determined up to 3 h after
injection, and both types of mice showed an increase in TNF
levels
starting at 30 min after injection and peaking at 60-90 min after LPS
injection (Fig. 3). The TNF
levels in control mice were significantly (p < 0.05)
lower than in the apoE-deficient mice, indicating that the presence of
apoE in the serum significantly protects the mice against the effects
of LPS.
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Fig. 3.
TNF levels of
ApoE-deficient and control mice. ApoE-deficient (
) and control
mice (
) were injected intravenously with LPS (25 µg/kg). At the
indicated time points, blood samples were taken and TNF
levels were
determined by ELISA. Data indicate the mean ± S.E.
(n = 5), asterisk indicates a significant
difference between LPS-treated apoE-deficient mice and LPS-treated
control mice (p < 0.05).
View larger version (19K):
[in a new window]
Fig. 4.
Effect of LPS on the serum levels of
endogenous apoE and total cholesterol. A and
B, mice were injected with PBS ( ) or LPS (
). At the
indicated times, blood samples were taken from the tail vein and
allowed to clot for 30 min. Serum was assayed for its content of apoE
(A) using a mouse apoE-specific ELISA and total cholesterol
(B). C, fasted apoE-deficient
(E(
/
)) and wild-type (E (+/+)) mice were
injected with PBS (control) or LPS. Blood samples were taken
before (time 0, open bars) and 12 h (12, closed
bars) after injection, and the sera were assayed for their apoE
content. C inset, Western blot analysis of the
serum proteins. Using a rabbit anti-mouse apoE antibody, apoE could be
detected as a single 34-kDa protein. Values are the mean ± S.E.
of three experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
levels was observed. This is
surprising because apoE-deficient mice have 8-fold higher cholesterol
levels than control mice, but the absence of apoE from the lipoproteins
apparently leads to an inability to neutralize LPS. These data are in
agreement with very recent data of de Bont et al. (45), who
showed that apoE-deficient mice produce significantly more TNF
in
response to LPS than control mice, and the mortality after injection of LPS was significantly higher in apoE-deficient mice than in control mice. The levels of IL-1 and IL-6 however were similar in
apoE-deficient and control mice in response to LPS.
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ACKNOWLEDGEMENT |
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We thank Dr. N. Pearce of SmithKline Beecham Pharmaceuticals, Harlow, UK for kindly providing us with the apoE antibody.
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FOOTNOTES |
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* This work was supported by Medical Sciences Grant 902-23-139 from The Netherlands Organization for Scientific Research, Council for Medical Research.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.
§ These authors contributed equally to this work.
¶ Present address: Radiation Genetics and Chemical Mutagenesis, University of Leiden, Sylvius Laboratories, P. O. Box 9503, 2300 RA Leiden, The Netherlands.
To whom correspondence should be addressed. Tel.: 31 715276040;
Fax: 31 715276032; E-mail: j.kuiper@lacdr.leidenuniv.nl.
Published, JBC Papers in Press, January 2, 2001, DOI 10.1074/jbc.M009915200
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ABBREVIATIONS |
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The abbreviations used are: LPS, lipopolysaccharide; apoE, apolipoprotein E; TNF, tumor necrosis factor; i.v., intravenous; PBS, phosphate-buffered saline; ELISA, enzyme-linked immunosorbent assay; IL, interleukin.
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