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INTRODUCTION |
We previously reported that high density lipoprotein
(HDL)1-associated enzymes can
protect LDL against oxidation by aortic wall cells (1, 2). However, we
also reported that during an acute phase response (APR) in both humans
and rabbits, HDL lost these protective enzymes and was converted from
an anti-inflammatory to a pro-inflammatory particle (3). In other
studies we observed that feeding an atherogenic diet to mice
genetically susceptible to atherosclerosis resulted in changes similar
to those seen in the APR. Both the plasma and hepatic expression of two
HDL- associated proteins, apolipoprotein (apo) J and paraoxonase (PON),
were altered. ApoJ, a marker of the acute phase response, was
increased, but there was a marked decline in PON expression and
activity (3, 4). These changes were not seen in mice genetically
resistant to atherosclerosis (4). Injection of oxidized phospholipids responsible for the biological activity of mildly oxidized LDL into
susceptible mice resulted in a similar pattern of expression, i.e. apoJ was increased, whereas PON was reduced (4).
Previously, we had demonstrated that injection of mildly oxidized LDL
but not native LDL induced the expression of JE, the murine homologue for monocyte chemoattractant protein-1 (MCP-1) (5). We also had
reported that an atherogenic diet induced conjugated dienes and the
expression of several inflammatory and oxidative stress-responsive genes in the liver of C57BL/6J mice (6). More recently we reported that
an atherogenic diet when fed to mice induced the hepatic formation of
the oxidized phospholipids that are responsible for the biologic
activity of mildly oxidized LDL (7).
Feingold and associates (8, 9) observed an increase in hepatic apoJ
expression and a decrease in hepatic PON expression in Syrian hamsters
with inflammatory cytokines, specifically TNF-
and interleukin
(IL)-1. Another principal mediator of the APR, IL-6, is also known to
affect the hepatic synthesis of a number of apolipoproteins and acute
phase reactants (10), in vivo and in vitro (11),
however its role in artery wall metabolism is less well understood.
Studies from the literature support both a positive (12) and negative
role (13) for IL-6 in the development of atherosclerosis. The data
reported here demonstrate that oxidized phospholipids found in mildly
oxidized LDL act to acutely alter the expression of PON and apoJ, at
least in part, through the inflammatory cytokine, IL-6. However, the
mechanisms by which oxidized phospholipids alter PON and apoJ
expression in the liver are different from those required to induce
MCP-1, and the short-term regulation of PON differs from long term regulation.
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EXPERIMENTAL PROCEDURES |
Materials--
Tissue culture materials and other reagents were
obtained from sources described previously (1, 4, 11). PAPC was
obtained from Sigma or from Avanti Polar Lipids (Alabaster, AL).
Recombinant IL-6, antibodies to IL-6, and ELISA kits to measure murine
IL-6 and human MCP-1 levels were obtained from
BIOSOURCE (Thousand Oaks, CA).
Cell Culture--
HepG-2 cells were propagated in 10% fetal
bovine serum in Eagle's modified essential medium supplemented with
1% sodium pyruvate, 1% penicillin-streptomycin-glutamine, 1%
nonessential amino acids, and 0.05% Fungizone in tissue culture flasks
treated with 0.1-0.5% gelatin at 37 °C overnight. HepG-2 cells
were seeded at a density of 1 × 105
cells/cm2 and allowed to reach a confluence of 80-85% by
the time of the experiment.
Human aortic endothelial cells were isolated from aortic specimens
obtained from donor hearts. Human aortic endothelial cells were
subsequently seeded at 2 × 105 cells/cm2
and were allowed to grow forming a complete monolayer of confluent endothelial cells in 2 days. At the time of experiment, the cells were
washed and transferred to Ham's F-10 medium containing 10% LPDS for
1 h to equilibrate the cells. The cells were subsequently washed,
and fresh media containing the experimental additions were added.
Mice--
Female C57BL/6J and C57BL/6J mice genetically lacking
IL-6 (IL-6
/
), 8-10 weeks of age, were purchased from the Jackson Laboratory, Bar Harbor, ME. Mice were bled under anesthesia by retro-orbital puncture, in accordance with protocols approved by the
UCLA Animal Research Protection Committee.
Diets--
Purina chow diet (Ralston Purina Co., St. Louis, MO)
or an atherogenic diet of 15% fat, 1.25% cholesterol, and 0.5%
cholic acid (Teklad, Madison, WI) were used in these studies.
Histological Analysis--
The heart, including the aortic root,
was dissected out and washed once in phosphate-buffered saline. The
basal portion of the heart and aortic root were embedded in OCT
compound (Miles Inc., Elkhart, IN) and frozen on dry ice. Serial
10-µm-thick cryosections of the heart tissue, covering the area
between the appearance of the mitral valves to the disappearance of the
aortic valves, were prepared. Every third section was collected on a
poly-L-lysine-coated slide. All sections were stained with
Oil Red O to assess aortic fatty streak development.
Preparation of Oxidized Lipids--
OX-PAPC,
1-palmitoyl-2-(5-oxovaleryl)-sn-glycero-3-phosphorylcholine
(m/z 594), and
1-palmitoyl-2-glutaryl-sn-glycero-3-phosphorylcholine (m/z 610), were prepared as described previously (12).
Quantitation of Gene Expression--
Total RNA was extracted
from the HepG2 cells according to the technique of Chomczyski and
Sacchi (13). Northern blot analysis was used to quantitate the mRNA
levels of apoJ and PON. For each sample, 15 µg of RNA were
electrophoresed on formaldehyde, 1% agarose gels and transferred to
20× SSC equilibrated Hybond ECLTM nitrocellulose membrane. Membranes
were hybridized following UV cross-linking and washed at a high
stringency (65 °C, 0.1× SSC). The apoJ probe had a sequence of:
5'-CAGTCCACAGACAAGATCTCCTGGCACTTTTCACACTGGC-3'. The PON probe had a
sequence of: 5'-attatcttcATCTGTGAATGTGCTAATCCCATGAGGGTTAAATG-3' (14). The blots were also hybridized using a cDNA probe for 18 or
28 S ribosomal RNA, or a cDNA probe for glyceraldehyde-3-phosphate dehydrogenase to normalize the quantities of RNA loaded into the gel lanes.
PON Activity--
Plasma samples were assayed for PON activity
using paraoxon as substrate (15). The cuvette contained 1.0 mM paraoxon in 20 mM Tris-HCl, pH 8.0. The
reaction was initiated by the addition of the plasma sample and the
increase in the absorbance at 405 nm was recorded over a 90-s period.
Blanks were included to correct for the spontaneous hydrolysis of
paraoxon. Enzymatic activity was calculated from the molar extinction
coefficient 1310 M
1
cm
1. A unit of PON activity is defined as 1 nmol of 4-nitrophenol formed per minute under the above assay
conditions (15).
Other Procedures--
All reagents and lipoproteins used in
these studies were analyzed for the presence of lipopolysaccharide
(LPS) contamination using a Limulus Amebocyte Lysate kit (BioWhittaker)
and were found to contain
0.05 pg/µg. For the detection of apoJ in
mouse plasma, an ELISA assay was used employing a clusterin/apoJ
primary antibody, purchased from Chemicon (Temecula, CA). The secondary
antibody was an anti-goat IgG from Vector Laboratories (Burlingame,
CA). Statistical analyses were carried out first using model I analysis of variance to determine whether differences existed among the group
means, followed by a Student's t distribution to identify the significantly different means. Cholesterol concentrations of HDL
were determined using a Cholesterol-20 kit from Sigma.
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RESULTS |
Effects of Oxidized Lipids on PON and ApoJ mRNA Expression in
HepG2 Cells--
In previous studies we had observed that HepG2 cells
exposed to LDL that had been oxidized by cocultures of smooth muscle cells and endothelial cells (CM-LDL) for 16 h demonstrated a
decrease in mRNA for PON and an increase in apoJ mRNA (4). To
determine whether specific oxidized
arachidonic acid-containing phospholipids would produce similar
results, the experiments shown in Figs. 1 and
2 were carried out. HepG2 cells incubated
for 16 h in medium alone or in medium containing 100 µg/ml
nonoxidized phospholipid PAPC showed no differences with respect to
the levels of expression of PON mRNA (Fig. 1). However, in HepG2
cells exposed to either 100 µg/ml OX-PAPC or 10 µg/ml amounts of
two synthetic oxidized phospholipids having m/z of 594 and
610, respectively (10), a marked decrease in PON mRNA expression
was observed. In addition, as demonstrated in Fig. 2, whereas no
significant differences were seen in HepG2 cells with respect to apoJ
mRNA expression, when the cells were treated with PAPC, treatment
of cells with the oxidized phospholipids resulted in a small but
significant 25% increase in mRNA expression.

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Fig. 1.
Effect of oxidized lipids on PON mRNA
expression in HepG2 cells. HepG2 cells were either untreated in
medium alone, or treated with either 100 µg/ml
1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphoryl choline
alone (PAPC), 100 µg/ml oxidized PAPC
(OX-PAPC), or 10 µg/ml oxidized phospholipids having a
m/z of 594 or 610. Total RNA was prepared for Northern blot
analysis as described under "Experimental Procedures." Values shown
are the mean ± S.D. of densitometric scans from three separate
experiments normalized for 28 S ribosomal RNA expression.
Asterisks indicate significant difference at the level of
p < 0.05, between medium alone and each
treatment.
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Fig. 2.
Effect of oxidized lipids on apoJ mRNA
expression in HepG2 cells. HepG2 cells were either untreated in
medium alone (Media), or treated with either 100 µg/ml
1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphoryl choline
alone (PAPC), 100 µg/ml each of two separate preparations
of oxidized PAPC (OX-PAPC), or 10 µg/ml oxidized
phospholipids having a m/z of 594 or 610. Total RNA was
prepared for Northern blot analysis as described under "Experimental
Procedures." Values shown are the mean ± S.D. of densitometric
scans from three separate experiments normalized for 28 S ribosomal RNA
expression. Asterisks indicate significant difference at the
level of p < 0.05, between medium alone and each
treatment.
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Role of IL-6 in Mediating the Effects of Oxidized Phospholipids on
HepG2 Cells--
One of the principal mediators of both acute and
chronic inflammatory responses in many diseases is IL-6. To test if
this cytokine might play a role in the inflammatory responses elicited by the oxidized phospholipids, the experiment shown in Fig.
3 was carried out. HepG2 cells were
incubated in tissue culture medium alone, in medium containing 100 µg/ml OX-PAPC, in medium containing 1 µg/ml neutralizing antibody
to IL-6 alone, or in medium containing 100 µg/ml OX-PAPC but also
containing 1 µg/ml neutralizing antibody to IL-6. Fig. 3 illustrates
that, similar to what was seen in Fig. 2, OX-PAPC inhibited the
expression of PON mRNA in HepG2 cells. However, in the presence of
neutralizing antibody to IL-6, there was a marked blunting of the
decrease in PON mRNA expression in response to OX-PAPC. Likewise,
as shown in Fig. 3B, neutralizing antibodies to IL-6 blocked
the stimulation of apoJ mRNA by OX-PAPC. Medium containing
neutralizing antibodies to IL-6 had no significant effect on either PON
or apoJ mRNA expression. The differences in apoJ or PON mRNA in
media alone,
-IL-6 in media, or OX +
-IL-6 were not statistically
significant. Moreover, irrelevant antibodies had no effect on either
PON or apoJ mRNA expression (data not shown). To directly test the
effects of IL-6, the experiment in Fig. 4
was conducted. HepG2 cells were incubated in medium alone, or in medium
containing a dose of recombinant IL-6 shown to be active in
vitro (100 ng/ml) (11) for 16 h and then mRNA levels for
PON and apoJ measured. IL-6 was effective in mimicking the effects of
OX-PAPC on PON and apoJ mRNA expression, causing a reduction in PON
mRNA and a stimulation of apoJ mRNA

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Fig. 3.
Effect of antibody to IL-6 on PON and apoJ
mRNA expression in HepG2 cells. HepG2 cells were either
untreated in medium alone (Media), or treated in medium with
either 100 µg/ml oxidized
1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphoryl choline
alone (OX), 1 µg/ml neutralizing antibody to IL-6 alone
( -IL-6), or 100 µg/ml oxidized
PAPC in the presence of 1 µg/ml neutralizing antibody to IL-6
(OX + -IL-6). Total RNA was prepared for
Northern blot analysis as described under "Experimental
Procedures." Values shown are the mean ± S.D. of densitometric
scans from three separate experiments normalized for 28 S ribosomal RNA
expression. Asterisks indicate significant difference at the
level of p < 0.05, between medium alone and each
treatment.
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Fig. 4.
Effect of IL-6 on PON and apoJ mRNA
expression in HepG2 cells. HepG2 cells were either untreated in
medium alone (Media), or treated with 100 ng/ml recombinant
IL-6 (IL-6). Total RNA was prepared for Northern
blot analysis as described under "Experimental Procedures." Values
shown are the mean ± S.D. of densitometric scans from three
separate experiments normalized for 18 S ribosomal RNA expression.
Asterisks indicate significant difference at the level of
p < 0.05, between medium alone and each
treatment.
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Absence of IL-6 Results in an Altered Response to Oxidized
Lipids--
We had previously demonstrated that injection of OX-PAPC
into C57BL/6J mice resulted in a marked reduction in plasma PON
activity (4). If IL-6 has a role in mediating the effects of oxidized lipids on altering apoJ levels and PON activity, then one might predict
that, in the absence of IL-6, this response would be diminished. We
therefore used IL-6 knockout mice (IL-6
/
) bred onto a C57BL/6J background to test the effects of OX-PAPC on altering plasma apoJ levels and PON activity in these mice. Fig.
5A shows that, compared with
uninjected mice or mice receiving an intravenous injection of saline or
PAPC, injection of OX-PAPC into the wild type strain, C57BL/6J mice
(WT) resulted in a marked decrease in plasma PON activity. Moreover,
this same concentration of OX-PAPC resulted in an increase in plasma
apoJ levels (Fig. 5B). When plasma levels of IL-6 were
measured by ELISA in WT mice 16 h after OX-PAPC injection, they
were found to be elevated 5-fold compared with saline or PAPC-treated
mice (data not shown). In contrast, neither plasma apoJ levels nor
plasma PON activity was altered in IL-6
/
mice after injection of
OX-PAPC compared with plasma PON activities in IL-6
/
that were
either untreated, or treated with saline or PAPC. Both strains,
however, responded to an injection of LPS, demonstrating an increase in
apoJ and a reduction in PON activity. Injecting a lower dose of 2.5 µg of LPS resulted in similar but smaller changes in both apoJ and
PON activity (data not shown). Expression of mRNA for both PON and
apoJ reflected the plasma PON profiles in the mice (data not shown).
These results would suggest that an IL-6-independent pathway is
involved in the action of LPS on apoJ and PON.

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Fig. 5.
The effects of IL-6 deficiency on the
response to OX-PAPC. Groups of five female C57BL/6J (WT,
open bars) or IL-6-deficient mice
(IL-6 / , solid bars) were either
uninjected (UN), or injected via tail vein with either 100 µl of saline (SA), 500 µg of PAPC (PA) or
OX-PAPC (OX) in 100 µl of saline or with 25 µg of
lipopolysaccharide in 100 µl of saline (LPS). After
16 h blood was removed under anesthesia for the determination of
PON activity and apoJ levels in the plasma, and the animals were
sacrificed to remove livers for total RNA isolation. Panel
A, PON activity; panel B, apoJ levels. Values shown are
the mean ± S.D. Asterisks indicate significant
difference at the level of p < 0.05.
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Since IL-6
/
mice did not exhibit the OX-PAPC-induced reduction in
plasma PON activity observed in C57BL/6J mice, we tested the hypothesis
that IL-6 may have a role in modulating the aortic response to oxidized
phospholipids. To first address this question, we examined the effects
of OX-PAPC on the production of a key chemokine by endothelial cells,
MCP-1, in the absence of IL-6, by using neutralizing antibody to this
cytokine. As shown in Fig. 6, compared
with endothelial cells incubated in the presence of PAPC, the addition
of OX-PAPC resulted in an increase in MCP-1 in the medium similar to
the levels seen with LPS and IL-6 alone. However, neutralizing antibody
to IL-6, which reduced the level of MCP-1 induced by IL-6 alone (data
not shown), had no significant effect on the induction of MCP-1 by
OX-PAPC. This raised two questions. First, is the role of IL-6 in the
response to oxidized phospholipids unique to the liver cell, and
second, are the mechanisms responsible for inducing apoJ and PON
different from those for MCP-1? Fig. 7
shows that, in contrast to the effects of antibody to IL-6 on OX-PAPC-mediated alterations in PON and apoJ in HepG2 cells (Fig. 3),
antibody to IL-6 had no effect on OX-PAPC-mediated MCP-1 expression in
HepG2 cells. This would suggest that, indeed, the mechanisms mediating
the action of oxidized phospholipids on PON and apoJ are different from
those required to induce MCP-1.

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Fig. 6.
The effects of antibody to IL-6 on
OX-PAPC-induced MCP-1 secretion by human aortic endothelial cells.
Human aortic endothelial cells were cultured and allowed to grow
forming a confluent monolayer. At the time of experiment, the cells
were washed and transferred to Ham's F-10 medium containing 10% LPDS
for 16 h to equilibrate the cells. The cells were subsequently
washed, and fresh media containing the following experimental additions
were added for 4 h: no additions (N/A), 25 µg/ml PAPC
(PAPC), 25 µg/ml OX-PAPC (OX), 25 µg/ml OX-PAPC in the presence of 1 µg/ml neutralizing antibody to
IL-6 (OX + -IL6), 25 µg/ml OX-PAPC in the
presence of 1 µg/ml irrelevant antibody (OX + IR), 100 ng/ml recombinant IL-6 (IL-6), or 100 ng/ml LPS
(LPS). Media were collected and samples assayed for MCP-1
concentrations by ELISA as described under "Experimental
Procedures." Values shown are the mean ± S.D. from three
separate experiments. Asterisks indicate significant
difference at the level of p < 0.05.
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Fig. 7.
The effects of antibody to IL-6 on
OX-PAPC-induced MCP-1 mRNA expression in HepG2 cells. HepG2
cells were cultured and allowed to grow to 85% confluence. At the time
of experiment, the cells were washed and transferred to Ham's F-10
medium containing 10% LPDS for 16 h to equilibrate the cells. The
cells were subsequently washed, and fresh media containing the
following experimental additions were added for 4 h: no additions
(N/A), 25 µg/ml PAPC (PAPC), 25 µg/ml OX-PAPC
(OX-PAPC), 100 ng/ml recombinant IL-6
(IL-6), 100 ng/ml LPS (LPS), 25 µg/ml OX-PAPC in the presence of 1 µg/ml neutralizing antibody to
IL-6 (OX + -IL-6), and 25 µg/ml OX-PAPC in
the presence of 1 µg/ml irrelevant antibody (OX + IR).
Media were collected and samples assayed for MCP-1 concentrations by
ELISA as described under "Experimental Procedures." Values shown
are the mean ± S.D. from three separate experiments.
Asterisks indicate significant difference at the level of
p < 0.05.
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These results suggested that, acutely, IL-6 plays a role in PON
regulation. However, would IL-6 also play a role in the regulation of
PON as well as in fatty streak development in the face of a long
term oxidative stress? To test this, we fed WT and IL-6
/
mice
an atherogenic diet for 15 weeks and then measured both plasma PON
activity and liver mRNA levels as well as the extent of aortic fatty streak formation. As shown in Fig.
8A, PON activity decreased in
both WT and IL-6
/
fed the atherogenic diet, compared with mice on
chow. Changes in PON mRNA essentially mirrored changes in activity
(Fig. 8B). The extent to which the PON activity decreased in
the IL-6
/
on the atherogenic diet was less than in the WT; however, these differences were small and there was a greater than 80%
reduction in both groups of animals. The small differences in activity
could not be detected as differences in mRNA expression between WT
and IL-6
/
. Despite similar low PON activity and mRNA levels,
IL-6
/
mice were found to have significantly greater areas of fatty
streak involvement in their aortas than did WT mice (Fig.
9). Neither strain developed lesions on a
chow diet (data not shown). These results would suggest that, under the conditions employed here (the atherogenic diet), relatively small changes in PON activity were overshadowed by other events in the artery
wall.

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Fig. 8.
The effects of an atherogenic diet on PON
activity (A) and mRNA (B) in IL-6-deficient
mice. Groups of 10 female C57BL/6J (WT) or
IL-6-deficient mice (IL-6 / ) mice were fed an
atherogenic diet of 15% fat, 1.25% cholesterol, and 0.5% cholic acid
for 15 weeks (ATH, solid bars). At the end
of this time, blood was removed under anesthesia for the determination
of PON activity and the animals were sacrificed to remove livers for
total RNA isolation. One group each of WT and IL-6 / on chow were
also sacrificed for controls (CHOW, open bars). Values shown
are the mean ± S.D. Asterisks indicate significant
difference at the level of p < 0.05 for chow
versus ATH. Plus symbol indicates
significant difference from WT at the level of p < 0.05 for animals on the atherogenic diet.
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Fig. 9.
The effects of IL-6 deficiency on aortic
fatty streak formation. Groups of 10 female C57BL/6J mice
(WT, open bars) or IL-6-deficient mice
(IL-6 / , solid bars) were fed an
atherogenic diet of 15% fat, 1.25% cholesterol, and 0.5% cholic acid
for 15 weeks. At the end of this time, mice were anesthetized and
sacrificed to remove hearts and aortas, and lesion analysis was carried
out as described under "Experimental Procedures." Values shown are
the mean ± S.D. Asterisk indicates significant
difference at the level of p < 0.05. GAPDH,
glyceraldehyde-3-phosphate dehydrogenase.
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DISCUSSION |
Our previous results in animal models demonstrated that the
decrease in the PON/apoJ ratio induced by administration of oxidized phospholipids, by a high fat atherogenic diet, or during an APR contributed to the formation of HDL that were no longer
anti-inflammatory in nature but were in fact pro-inflammatory (3, 4).
In the present study, we have demonstrated that specific oxidized
phospholipids found in MM-LDL affected the gene expression of two
HDL-associated proteins, PON and apoJ, when incubated with cultured
HepG2 cells. This response was blocked by antibodies to IL-6. HepG2
cells treated directly with IL-6 in the absence of oxidized
phospholipids showed the same pattern of response for PON and apoJ
mRNA expression.
Others have noted effects of cytokines on hepatic lipoprotein
synthesis, suggesting a link between the acute phase response and the
development of atherosclerosis. Memon and co-workers (14) observed that
both IL-1 and tumor necrosis factor increased serine palmitoyltransferase, the rate-limiting step in sphingolipid synthesis, resulting in an altered lipoprotein structure. Song et al.
(15) observed that IL-1
and TNF-
suppressed apoA-I expression in HepG2 cells but not expression of apoE. Feingold and co-workers (9)
demonstrated that HepG2 cells treated with the other major APR
cytokines, TNF-
and IL-1, showed a decrease in PON mRNA levels. Moreover, other changes in lipoprotein metabolism that could
potentially influence the development of atherosclerosis are increases
in serum triglyceride levels, an increase in small dense LDL, changes in the composition of HDL, including the activities of its enzymes other than PON, particularly lecithin cholesterol acyltransferase and
cholesteryl ester transfer protein (16, 17).
We previously demonstrated that PON is capable of abolishing the
bioactivity of mildly oxidized LDL and that of OX-PAPC (1). Alterations
in plasma PON activity have been reported in several pathological
conditions. Abbott and colleagues (18) and Mackness and Durrington (19)
have reported low PON activity in diabetes and that a low serum PON
activity was associated with increased susceptibility to
atherosclerosis (20). ApoJ, also known as clusterin, is a glycoprotein
that is present in a subpopulation of HDL together with PON and apoA-I
(21, 22). It has been reported to have roles in complement regulation
and prevention of cytolysis (23), lipid transport (24), apoptosis (24), and membrane protection at fluid-tissue interfaces where it is expressed (25). HepG2 cells secrete apoJ as a lipoprotein, indicating its ability to be associated with lipid (26). One could postulate that
the association of apoJ and PON in the plasma, and their reciprocal
regulation by the liver, might act as a coordinated protection during
inflammatory conditions.
These studies support the notion that, during an acute oxidative
stress, the effects of oxidized phospholipids may be mediated by
inflammatory cytokines. The action of IL-6 may be via its activation of
gp130, a transmembrane glycoprotein involved in many cytokine-mediated cellular responses (27). Additional support for a role of oxidant stress on cytokine activation comes from the work of Eugui et al. (28). These investigators found that antioxidants such as butylated hydroxyanisole, tetrahydropapaveroline, nordihydroguiauretic acid, and 10,11-dihydroxyaporphine inhibited the
production of IL-6 as well as TNF-
and IL-1
by human peripheral
blood monocytes, suggesting that suppressing oxidation can limit the
inflammatory response.
In the present study, injection of OX-PAPC into C57BL/6J mice resulted
in a dramatic reduction in plasma PON activity after 16 h.
However, injection of OX-PAPC into IL-6
/
mice had no such effect.
Others have found that, in IL-6-deficient mice, IL-6 was absolutely
required for the transcriptional induction of hepatic APR genes
following injection of turpentine oil (29).
Tissue levels of oxidized phospholipids were not measured in the
in vivo studies reported here. Therefore, we cannot
determine the role of oxidized lipids in vivo directly from
these studies. For the purpose of discussion, we assume that these
lipids were formed as they were in our previous studies (7). To
determine whether the cells of the artery wall behave differently with
respect to IL-6 in response to oxidized phospholipids, we incubated
aortic endothelial cells with OX-PAPC in the presence or absence of
neutralizing antibody to IL-6 and measured the release of MCP-1 into
the tissue culture medium. We had previously demonstrated, using
neutralizing antibody to MCP-1, that >95% of the monocyte chemotactic
activity in an artery wall coculture system was due to the presence of MCP-1 (30). Although IL-6 itself increased MCP-1 secretion from endothelial cells, we found that neutralizing antibody to IL-6 did not
alter OX-PAPC-induced MCP-1 secretion by endothelial cells (Fig. 6).
Moreover, neutralizing antibody to IL-6 had no effect on
OX-PAPC-induced MCP-1 mRNA expression in HepG2 cells (Fig. 7). We
reasoned that if indeed IL-6 had a role in mediating the action of
oxidized lipids in the liver, then this cytokine might also have an
influence on the events in the artery wall leading to the development
of fatty streaks. To test this hypothesis, IL-6
/
mice were fed an
atherogenic diet for 15 weeks and compared with their background
strain, C57BL/6J mice, for the extent of lesion development. To our
surprise, IL-6
/
mice were found to have significantly greater
areas of fatty streak involvement in their aortas than did WT mice
(Fig. 9) despite the small but significant changes in PON activity.
Sukovich (13) recently reported in abstract form that there was no
difference in plaque surface area in apoE/IL-6 double knockout mice
compared with apoE
/
mice, but that the plaques were less fibrotic.
IL-6
/
mice treated with lipopolysaccharide had a 3-fold greater
production of TNF-
than in wild type controls (31). In other
studies, IL-6
/
mice had 30-50% of the circulating plasma levels
of TNF-
and IL-1
(32). In our study, the decrease in PON activity
in IL-6
/
mice was slightly although significantly less than in the
WT mice on the atherogenic diet. However, there was a substantial reduction in PON activity in both the IL-6
/
and the WT mice fed
the atherogenic diet compared with the mice fed a chow diet. The
difference between the results in Figs. 5 and 8 may result from a
difference in the magnitude and duration of the oxidative stress (15 weeks on the atherogenic diet). In the chronic situation, PON activity
was decreased despite the absence of IL-6. This suggests that IL-6 may
be critical to short term regulation of PON, but not to long term
regulation. If these results can be extrapolated to humans, it might
also suggest that short term regulation of PON activity mediated by
IL-6, together with the IL-6 induction of MCP-1 in endothelial cells
(Fig. 6), might exacerbate the inflammatory reaction in an established
atherosclerotic lesion.
A number of epidemiological studies have observed a link between acute
inflammation and cardiovascular diseases. Changes in the levels of
plasma C-reactive protein have been linked with an increased
cardiovascular risk (33). Concentrations of C-reactive protein and
serum amyloid A have also been shown to increase acutely after
cholescystectomy, returning to normal within 2 weeks (34). Plasma
concentrations of IL-6 are elevated in a number of inflammatory states
as diverse as rheumatoid arthritis (35), lymphoma (36), orthopedic
surgery (37), and cardiovascular disease (38). Since IL-6 has been
shown to induce the serum amyloid A promoter (39), it is
possible that factors that can elevate IL-6 levels, such as oxidized
phospholipids, may be an initial step in the alterations observed
during an acute phase response. In mice, inoculation with murine
cytomegalovirus increased LDL-derived cholesterol and resulted in a
greater incidence of early atherosclerotic lesions (40).
Chlamydia pneumoniae has been repeatedly demonstrated to be
associated with the incidence of atherosclerosis by both serology and
the demonstration of the organism in atherosclerotic lesions (41). An
intriguing relationship has also been made between acute respiratory
infections and the risk of first time acute myocardial infarction (42).
Another study has shown that the presence of an inflammatory response
has a prognostic value in patients with unstable angina and may predict
the long term risk of cardiovascular events (43). Finally, there are
results of two preliminary antibiotic treatment trials using
azithromycin for the secondary prevention of cardiovascular disease
(44). It is possible that the nonspecific immune response to these
infectious agents, the APR, may play a role, and may be a mechanism
linking infections with chronic inflammatory processes. Work in this
laboratory is currently focused on the specific initiators of the
oxidant stress-mediated development of early atherosclerotic lesions, and investigating further the relationship between lipid oxidation and
the APR.