Acute inflammation increases selective uptake of HDL cholesteryl esters into adrenals of mice overexpressing human sPLA2
Uwe J. F. Tietge,1
Cyrille Maugeais,1
Willliam Cain,2 and
Daniel J. Rader1
1Department of Medicine, University of
Pennsylvania, Philadelphia, Pennsylvania 19104; and
2Department of Biology, University of Delaware,
Newark, Delaware 19716
Submitted 30 December 2002
; accepted in final form 18 February 2003
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ABSTRACT
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The acute-phase protein secretory phospholipase A2 (sPLA2)
influences the metabolism of high-density lipoproteins (HDL). The adrenals are
known to utilize HDL cholesterol as a source of sterols. The aim of the
present study was to test the hypothesis that sPLA2 enhances the
selective uptake of HDL into the adrenals in response to acute inflammation as
a possible physiological role for the sPLA2-HDL interaction. Human
sPLA2-transgenic mice, in which sPLA2 expression is
upregulated by inflammatory stimuli, were used. Ten hours after induction of
the acute-phase response (APR) by injection of bacterial lipopolysaccharide
(LPS), plasma levels of HDL cholesterol decreased significantly in
sPLA2-transgenic mice (-18%, P < 0.05) but remained
unchanged in wild-type mice. The fractional catabolic rates of both
125I-labeled tyraminecellobiose (TC)-HDL and
[3H]cholesteryl ether increased significantly in the
sPLA2-transgenic mice after induction of the APR (0.18 ±
0.01 vs. 0.21 ± 0.01 pool/h, P < 0.05, and 0.31 ±
0.02 vs. 0.42 ± 0.05 pool/h, P < 0.05, respectively) but
remained unchanged in the wild-type mice (0.10 ± 0.01 vs. 0.22 ±
0.02 pool/h, respectively). After induction of the APR, in both groups HDL
holoparticle uptake by the liver was increased (P < 0.001).
sPLA2-transgenic mice had 2.4-fold higher selective uptake into the
adrenals after induction of the APR than wild-type mice (156 ± 6 vs. 65
± 5%/µg tissue protein, P < 0.001). In summary,
upregulation of sPLA2 expression during the APR specifically
increases the selective uptake of HDL cholesteryl ester into the adrenals.
These data suggest a novel metabolic role for sPLA2: modification
of HDL during the APR to promote increased adrenal uptake of HDL cholesteryl
ester to serve as source for steroid hormone synthesis.
apolipoproteins; lipoproteins; kinetics; acute-phase response; lipopolysaccharide
DURING THE ACUTE-PHASE RESPONSE (APR), plasma levels
of HDL cholesterol are markedly decreased
(3,
15). Among the acute-phase
proteins that might have an impact on plasma HDL cholesterol metabolism is the
secretory group IIa phospholipase A2 (sPLA2). The family
of phospholipase genes with a potential impact on lipoprotein metabolism is
growing, with the latest discovered being phosphatidylserine-specific
phospholipase A1 (PS-PLA1)
(1) and lipase H
(18). sPLA2 is a
14-kDa Ca2+-dependent enzyme that hydrolyzes phospholipids at the
sn-2 position (8).
sPLA2 levels in plasma increase dramatically upon acute
inflammation (27), and plasma
sPLA2 activity has been localized to the HDL fraction in humans
(9). We recently reported that
transgenic overexpression of sPLA2 in mice in the absence of
systemic inflammation has profound effects on HDL metabolism: mice
overexpressing human sPLA2 have significantly lower plasma levels
of HDL cholesterol due to increased catabolism of HDL apolipoprotein A-I
(apoA-I), as well as HDL cholesteryl esters (CEs)
(34). However, the
physiological significance of sPLA2 expression during acute
inflammation and its effects on HDL metabolism remain unclear.
Transgenic mice overexpressing human sPLA2 survive challenges
with both Staphylococcus aureus
(23) and Escherichia
coli (22) better than
wild-type mice, suggesting that sPLA2 somehow protects against
acute bacterial challenge. However, the mechanism(s) of this protection is
unknown. We hypothesized that the metabolic effects of sPLA2 on HDL
metabolism might be related to its protective effects. For example, it is well
established that human apoA-I-transgenic mice are relatively protected against
endotoxin challenge (24). In
acute inflammation, the need for adrenal steroid hormone production is
increased, and HDL-CEs are a source of cholesterol for steroid hormone
synthesis (28,
30,
33,
41,
43). In vitro studies
(6) have suggested that
sPLA2 modification of HDL makes the CE component of the particle
more accessible to selective uptake via the scavenger receptor class B type I
(SR-BI) receptor. In separate experiments, we found that hepatic
overexpression of SR-BI enhanced selective uptake of HDL-CE to a greater
extent in sPLA2/apoA-I-double transgenic mice than in
apoA-I-transgenic mice (Tietge UJF, unpublished data). Thus one possible
function of sPLA2 might be to enhance the uptake of HDL-CEs into
the adrenals to meet the requirements for increased steroid hormone
synthesis.
Therefore, the purpose of our study was to test the hypothesis that
sPLA2 induction during the APR increases the selective uptake of
HDL-CE into the adrenals in vivo. The human sPLA2-transgenic mouse
model we utilized in our previous study
(34) represents an ideal tool
for addressing this question, because the sPLA2 transgene
expression is controlled by its own promoter and thereby confers upregulated
sPLA2 expression in response to inflammatory stimuli
(23). Therefore, we used human
sPLA2-transgenic and C57BL/6 mice, which lack endogenous
sPLA2 (19), to
perform a series of HDL kinetic studies after induction of the APR with
bacterial lipopolysaccharide (LPS) to assess plasma catabolism and tissue
uptake of HDL apoproteins as well as HDL-CEs. Our data indicate that, in human
sPLA2-transgenic mice, upregulation of sPLA2 in response
to acute inflammation results in significantly increased selective uptake of
HDL-CE into the adrenals, suggesting a potentially important novel
physiological function of sPLA2.
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EXPERIMENTAL PROCEDURES
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Animal studies. The human group II sPLA2-transgenic
mice used in this study have been described previously
(13,
34). Briefly, a 6.2-kb
HindIII restriction fragment, containing all five exons and 1.6 kb of
nucleotides upstream from the RNA initiation site and 0.35 kb of nucleotides
downstream of the polyadenylation signal sequence of the human
sPLA2 gene, was used to generate transgenic mice by standard
procedures. The sPLA2-transgenic line has been backcrossed to the
C57BL/6 background. Nontransgenic littermates from further breeding of
sPLA2-transgenic mice with wild-type C57BL/6 mice (Jackson
Laboratory, Bar Harbor, ME) were used as controls. The animals were caged in
animal rooms with alternating 12-h periods of light (7 AM-7 PM) and dark (7
PM-7 AM) and with ad libitum access to water and mouse chow diet.
The APR was induced by intraperitoneal injection of 40 µg of LPS (E.
coli 0111:B4, Difco Laboratories, Detroit, MI)
(16) or sterile saline. Blood
was obtained by retroorbital bleeding before injection and 10 h after
injection for determination of plasma lipid and lipoprotein levels. At the
10-h time point, mice were injected with the radiotracers, and the HDL kinetic
studies were carried out as described below.
Plasma lipid and lipoprotein analysis. Mice were bled from the
retroorbital plexus by use of heparinized capillary tubes. Blood was drawn
into tubes containing 2 mM EDTA, 0.2% NaN3, and 1 mM benzamidine.
Aliquots were stored at -20°C until analysis. Plasma total cholesterol,
HDL cholesterol, triglycerides, phospholipids, and human apoA-I levels were
determined on a Cobas Fara (Roche Diagnostics Systems, Nutley, NJ) by use of
Sigma Diagnostics reagents (Sigma Diagnostics, St. Louis, MO).
Western blotting. Western blot analysis for human group II
sPLA2 was performed, as previously described
(34), with a monoclonal mouse
anti-human sPLA2 primary antibody at a concentration of 2 µg/ml
(Boehringer Mannheim, Mannheim, Germany) and the enhanced chemiluminescence
(ECL) detection system (Amersham, Arlington Heights, IL).
HDL metabolic studies. Autologous HDL was prepared from pooled
mouse plasma by sequential ultracentrifugation (density: 1.063 < d <
1.21). After extensive dialysis against sterile PBS containing 0.01% EDTA, one
part of the HDL was labeled with 125I-tyramine-cellobiose (TC), as
previously described (34).
Unbound iodine was removed by passing the solution over a Sephadex G25
desalting column (Amersham Pharmacia Biotech). Finally, the HDL was reisolated
by ultracentrifugation (density: 1.063 < d <1.21), extensively dialyzed,
sterile filtered, and stored at 4°C until use
(34). The other part of the
isolated mouse HDL was labeled with cholesteryl hexadecyl ether
(cholesteryl-1,2-3H, NEN Life Sciences Products) according to a
previously published methodology
(34). After labeling, the HDL
was reisolated by ultracentrifugation (density: 1.063 < d <1.21),
extensively dialyzed, sterile filtered, and stored at 4°C until
injection.
To assess the rates of plasma catabolism and to measure the organ uptake of
the different tracers, 1 µCi of the 125I-TC-HDL and 1 million
dpm of the [3H]CE-HDL were coinjected via the tail veins into mice
from the different experimental groups. Blood samples were drawn by
retroorbital bleeding at 5 min, 1 h, 3 h, 6 h, 11 h, and 24 h (
25 µl
at each time point). An aliquot of 10 µl of plasma from each time point was
counted using a Cobra II
-counter (Packard Instruments, Downers Grove,
IL). These data were used to generate the plasma disappearance curves for HDL
apolipoproteins. After
-counting, the same samples were extracted with
the method described by Dole
(7). The lipid phases were
harvested, and aliquots were counted on a scintillation counter (Beckman
LS6500; Beckman Instruments, Palo Alto, CA). From these data, plasma
disappearance curves for HDL-CEs were calculated. The recovery of
[3H]cholesteryl ether after lipid extraction was 97.5 ±
1.9%. Contamination of the extracted 3H-labeled lipids with
125I was <0.5%. Plasma decay curves for both tracers were
generated by dividing the plasma radioactivity at each time point by the
radioactivity at the initial 5-min time point after tracer injection.
Fractional catabolic rates (FCRs) were determined from the area under the
plasma disappearance curves fitted to a bicompartmental model by use of the
SAAM II program (2).
To measure the organ uptake of HDL apolipoproteins and HDL-CEs, mice were
anesthetized with an intraperitoneally injected ketamine-xylazine mixture and
perfused extensively with cold PBS by cardiac puncture
(34). Then liver, spleen,
kidney, and adrenals were harvested. First, radioactivity uptake
(125I) into the different organs was assessed by
-counting
to determine uptake of HDL apolipoproteins. Then, the different organs were
extracted using the Dole method, and the amount of [3H]cholesteryl
ether taken up into each organ was determined on a scintillation counter
(Beckman LS6500). Organ uptake for each tracer was expressed as a percentage
of the injected dose. The injected dose was calculated by multiplying the
initial plasma counts (5-min time point) with the estimated plasma volume
(3.5% of total body wt). Selective uptake into each organ was determined by
subtracting the percentage of the injected dose of 125I-HDL
recovered in each organ from the percentage of the injected dose of
[3H]HDL-CE and correcting the value for tissue weight. Values were
expressed as micrograms of tissue weight for each organ.
Statistical analysis. Values are presented as means ± SE
unless otherwise indicated. Results were analyzed by ANOVA and Student's
t-test using the GraphPad Prism Software (GraphPad, San Diego, CA).
Statistical significance for all comparisons was assigned at P <
0.05.
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RESULTS
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In sPLA2-transgenic mice, injection of saline did not result in
any detectable change in plasma sPLA2, whereas plasma
sPLA2 was increased 10 h after induction of the APR by injection of
LPS (Fig. 1). These data,
consistent with a previous report
(23), demonstrate that the
sPLA2 in these transgenic mice is upregulated by acute
inflammation. At baseline, sPLA2-transgenic mice had significantly
lower plasma levels of total cholesterol (61 ± 3 vs. 85 ± 7
mg/dl, P < 0.01, Fig.
2A and Table
1), HDL-cholesterol (HDL-C; 44 ± 3 vs. 64 ± 5 mg/dl,
P < 0.01, Fig.
2B, Table
1), and phospholipids (116 ± 9 vs. 179 ± 4 mg/dl,
P < 0.01, Fig.
2C and Table
1) compared with wild-type mice, consistent with our previous
report (34). In both
saline-injected groups, plasma lipid levels remained relatively unchanged
compared with baseline values (Fig. 2,
AC). After LPS challenge in wild-type
mice, plasma total and HDL-C levels did not change significantly compared with
baseline values (Fig. 2, A and
B), but plasma phospholipid levels decreased
significantly by 19 ± 10% (P < 0.05 compared with baseline
values, Fig. 2C). In
contrast, in sPLA2-transgenic mice, plasma levels of total
cholesterol and HDL-C decreased significantly after LPS injection compared
with baseline values (-12 ± 5%, P < 0.05,
Fig. 1A; -18 ±
6%, P < 0.01, Fig.
1B, respectively), and phospholipids also decreased by 11
± 6% (Fig.
1C, P < 0.05 compared with baseline values).
Thus absence of sPLA2 is associated with a lack of HDL response to
endotoxin, whereas expression of inducible sPLA2 confers
responsiveness to LPS injection with regard to reduction of HDL-C levels.

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Fig. 1. Western blot analysis for human secretory phospholipase A2
(sPLA2) in plasma from sPLA2-transgenic mice at baseline
(0 h) and 10 h after injection with either saline or LPS as indicated. Samples
were resolved by SDS-PAGE under nondenaturing conditions. Human
sPLA2 was visualized using a monoclonal mouse anti-human
sPLA2 antibody, as described in EXPERIMENTAL
PROCEDURES.
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Fig. 2. Percent change of plasma lipids and lipoproteins 10 h after either the
intraperitoneal injection of saline or the induction of the acute-phase
response (APR) by injection of LPS in human sPLA2-transgenic mice
and C57BL/6 controls. A: total cholesterol; B: HDL
cholesterol; C: phospholipids.
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Table 1. Plasma lipid and lipoprotein profiles in human
sPLA2-transgenic mice and wild-type nontransgenic
littermates
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To assess the metabolic basis for the changes in HDL, we performed a series
of HDL metabolism studies simultaneously tracing the catabolism of both HDL
apolipoproteins and HDL-CEs in wild-type and sPLA2-transgenic mice
in reponse to endotoxin injection. We determined the rate and sites of HDL
apolipoprotein catabolism by using 125I-TC-HDL as a tracer.
Saline-injected sPLA2-transgenic mice had significantly faster
plasma catabolism of HDL apolipoproteins compared with saline-injected
wild-type controls, (0.18 ± 0.01 vs. 0.10 ± 0.01 pool/h,
P < 0.001, Fig. 3, A
and B, and Table
2), consistent with our previous observations
(34). LPS injection had no
significant effect on the catabolic rate of HDL apolipoproteins in wild-type
control mice (Table 2). On the
other hand, in sPLA2-transgenic mice, the FCR of HDL
apolipoproteins increased significantly in response to LPS (0.21 ± 0.01
pool/h, P < 0.05, Fig.
3B and Table
2). These data demonstrate that upregulation of the
sPLA2 transgene is required to mediate the effect of LPS injection
on HDL apolipoprotein catabolism.
Use of the trapped ligand 125I-TC-HDL allowed us to determine
the tissue sites of HDL apolipoprotein uptake. Uptake of HDL apolipoproteins
into the liver was not significantly different between saline-injected
sPLA2-transgenic mice and wild-type controls. However, after LPS
administration, hepatic uptake of the 125I-TC-HDL tracer increased
in wild-type mice (18 ± 2 to 27 ± 2% of injected dose,
P < 0.001, Fig.
4A) and in sPLA2-transgenic mice (15 ±
3 to 33 ± 3% of injected dose, P < 0.001,
Fig. 4A). There was no
difference in HDL apolipoprotein uptake into the spleen between the
saline-injected groups of experimental mice. After induction of the APR, there
was a significant increase in HDL apolipoprotein uptake into the spleen in
wild-type (0.7 ± 0.2 to 2.3 ± 0.3% of injected dose, P
< 0.001, Fig. 4B)
and sPLA2-transgenic mice (1.0 ± 0.2 to 2.1 ± 0.3% of
injected dose, P < 0.01, Fig.
4B). HDL apolipoprotein catabolism by the kidneys was
significantly higher in saline-injected sPLA2-transgenic mice than
in the wild-type mice (8 ± 1 to 5 ± 1% of injected dose,
P < 0.001), as we previously reported
(34), but did not change
significantly in either group after injection with LPS
(Fig. 4C). Uptake of
HDL apolipoproteins into the adrenals was extremely low relative to liver and
kidney (Fig. 4D).

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Fig. 4. Tissue sites of catabolism of 125I-TC-labeled autologous HDL in
sPLA2-transgenic (tg) and control mice after the injection of
either saline or LPS. Labeled HDL was prepared as described in
EXPERIMENTAL PROCEDURES and injected via the tail vein into
sPLA2-transgenic and control mice 10 h after induction of the APR
or injection of saline. After 24 h, mice were killed and thoroughly perfused
with PBS, and the respective tissues were harvested. Uptake of radioactivity
into the respective organs was determined by -counting. Data represent
percentages of injected tracer dose recovered in liver (A), spleen
(B), kidneys (C), and adrenals (D). Statistically
significant differences (P < 0.05) are as assessed by independent
sample t-test: *between saline-injected controls and
LPS-injected mice, and #between sPLA2-transgenic and wild-type
mice.
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We also investigated the plasma catabolism of HDL-CE by using as a tracer
HDL labeled with [3H]cholesteryl ether in the same mice used for
the HDL apolipoprotein kinetic studies described above. Human
sPLA2-transgenic mice injected with saline had significantly faster
plasma catabolism of HDL-CE than saline-injected control mice (0.31 ±
0.02 vs. 0.22 ± 0.02 pool/h, P < 0.001, respectively;
Fig. 5, A and
B, and Table
2), consistent with our previous report
(34). LPS injection had no
significant effect on plasma catabolism of HDL-CE in wild-type mice
(Fig. 5A,
Table 2). However, in
sPLA2-transgenic mice, LPS injection resulted in a significant
increase in the FCR of HDL-CE (0.42 ± 0.05 pool/h, P <
0.05, Fig. 5B,
Table 2). This indicates that
upregulation of the sPLA2 transgene is required to mediate the
effects of LPS injection on HDL-CE catabolism.
The use of the nonhydrolyzable CE analog [3H]cholesteryl ether
enabled the determination of sites of tissue catabolism of HDL-CEs. LPS
injection significantly increased hepatic HDL-CE uptake in wild-type mice (44
± 3 to 52 ± 4% of injected dose, P < 0.05,
Fig. 6A); in
sPLA2-transgenic mice, tracer uptake into the liver was
nonsignificantly increased (70 ± 4 to 78 ± 4% of injected dose,
P = 0.06, Fig.
6A). Uptake of HDL-CE into the spleen was increased in
both groups of LPS-injected mice, although only in wild-type mice did this
increase reach the level of statistical significance (P < 0.05,
Fig. 6B). There were
no significant changes in the small amount of HDL-CE uptake into the kidneys
between the different experimental groups of mice
(Fig. 6C).
Saline-injected sPLA2-transgenic mice had significantly higher
uptake of HDL-CE into their adrenals compared with wild-type controls (0.82
± 0.06 vs. 0.51 ± 0.06% of injected dose, P < 0.001,
Fig. 6D), consistent
with our previous report (34).
HDL-CE uptake into the adrenals increased significantly in both LPS-injected
sPLA2-transgenic mice (1.43 ± 0.11% of injected dose,
P < 0.001, Fig.
6D) and LPS-injected wild-type mice (0.82 ± 0.06%
of injected dose, P < 0.001,
Fig. 6D).

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Fig. 6. Tissue sites of catabolism of [3H]cholesteryl ether-labeled
autologous HDL in sPLA2-transgenic and control mice after injection
of either saline or LPS. Labeled HDL was prepared as described in
EXPERIMENTAL PROCEDURES and injected via the tail vein into
sPLA2-transgenic and control mice 10 h after induction of APR or
injection of saline. After 24 h, mice were killed and thoroughly perfused with
PBS, and the respective tissues were harvested. Organs were extracted
according to the Dole method, and radioactivity in the lipid phase was
determined by scintillation counting. Data represent percentages of injected
tracer dose recovered in liver (A), spleen (B), kidneys
(C), and adrenals (D). Statistically significant differences
(P < 0.05) are as assessed by independent sample t-test:
*between saline-injected controls and LPS-injected mice; #between
sPLA2-transgenic and wild-type mice.
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Because the HDL apolipoprotein and HDL-CE kinetic experiments were carried
out in the same mice, we used both measurements to determine the selective
uptake of CE from HDL into respective organs in response to LPS injection.
sPLA2-transgenic mice injected with saline had significantly higher
HDL-CE selective uptake into the liver (34 ± 2 vs. 25 ± 3%/µg
tissue protein, P < 0.05, Fig.
7A) compared with saline-injected wild-type controls, but
LPS injection had no significant effect on hepatic selective uptake of HDL-CE
in either group (Fig.
7A). Likewise, there was little effect of LPS on
selective uptake of HDL-CE in the spleen in either group. In the kidneys,
there was significantly higher "negative selective uptake" (i.e.,
lipid-poor protein uptake) in saline-injected sPLA2-transgenic mice
compared with wild-type mice (-23 ± 2 vs. -17 ± 2%/µg,
P < 0.01, Fig.
7C), and this was relatively unaffected by LPS injection
(Fig. 7C). In the
adrenals, HDL-CE selective uptake after LPS injection was 2.4-fold higher in
sPLA2-transgenic mice compared with wild-type mice (156 ± 6
vs. 65 ± 5%/µg tissue protein, P < 0.001,
Fig. 7D).

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Fig. 7. Selective uptake of HDL cholesteryl ester into tissues of
sPLA2-transgenic and control mice after injection of either saline
or LPS. For calculation, data shown in Figs.
4 and
6 were used.
125I-TC-labeled HDL uptake (% of injected dose) was subtracted from
[3H]cholesteryl ether-labeled HDL uptake (% of injected dose) into
the respective organs [liver (A), spleen (B), kidneys
(C), and adrenals (D)] to calculate selective uptake, and
data were referred to as µg of tissue wt. Statistically significant
differences (P < 0.05) are as assessed by independent sample
t-test: *between saline-injected controls and LPS-injected
mice; #between sPLA2-transgenic and wild-type mice.
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DISCUSSION
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The results of this study demonstrate that upregulation of sPLA2
expression by endotoxin injection significantly enhances the plasma catabolism
of HDL, results in altered tissue uptake of HDL apolipoproteins and HDL-CE,
and specifically increases the selective uptake of HDL-CE into the adrenals,
suggesting a novel and important metabolic in vivo role of
sPLA2.
The line of sPLA2-transgenic mice used in the present study was
made with a genomic construct that results in relatively high baseline
sPLA2 expression but also preserves the inducibility of the
transgene in response to inflammatory stimuli
(23). This allowed us to study
the effects of acute inflammation on HDL metabolism in human
sPLA2-transgenic mice on a C57BL/6 genetic background compared with
wild-type C57BL/6 mice lacking the endogenous mouse sPLA2 enzyme
due to a frameshift mutation in the respective gene
(19). At baseline,
sPLA2-transgenic mice have lower plasma levels of HDL-C compared
with wild-type mice (34). The
underlying metabolic mechanism is a significantly increased rate of plasma
catabolism of HDL apolipoproteins as well as HDL-CE
(34), a finding that we
confirmed in the current studies. In these studies, induction of the APR in
wild-type C57BL/6 mice neither altered the steady-state plasma concentrations
of HDL-C nor affected the catabolic rates of HDL apolipoproteins or HDL-CE. In
contrast, in sPLA2-transgenic mice, induction of the APR resulted
in increased sPLA2 expression and a significant decrease in HDL-C
levels. The FCRs of both HDL apolipoproteins and HDL-CE increased
significantly in sPLA2-transgenic mice in response to LPS
injection. These results indicate that the presence of an inducible and
functional sPLA2 gene is required to mediate the effects of acute
inflammation on HDL metabolism in mice. In parallel and separate experiments,
we crossed human sPLA2-transgenic mice with human apoA-I-transgenic
mice and found, consistent with the present results, that upregulation of
sPLA2 expression was required for an effect of LPS on plasma HDL-C
and apoA-I levels (35).
There are three possible pathways for the cellular uptake of HDL-C:
1) holoparticle uptake of HDL, 2) selective uptake of HDL-C
mediated by the SR-BI receptor, and 3) transfer to LDL by CE transfer
protein (CETP) and uptake via the LDL receptor. The latter pathway is not
active in mice, because this species is lacking CETP. Holoparticle uptake of
HDL is a known mechanism for removal of HDL from the circulation
(11,
12,
32,
42). One possible receptor for
this process is cubilin (26),
which may have a role in the renal and placental uptake of HDL
(14,
20). In the liver, other
poorly characterized mechanisms appear to exist that mediate holoparticle
catabolism of HDL. Our data demonstrate that, during the APR, a substantial
increase in the hepatic uptake of HDL apolipoprotein and CE occurs, with
holoparticle uptake being the most likely mechanism. This process appears to
be relatively unaffected by sPLA2. Likewise, there was no
significant change in HDL apolipoprotein uptake by the kidneys in
sPLA2-transgenic mice after induction of the APR. Therefore, our
data suggest that sPLA2 has relatively little, if any, effect on
holoparticle HDL uptake by the liver or kidney.
SR-BI has been extensively studied as a receptor that mediates the
selective uptake of HDL-C (21,
36,
40). Liver and steroidogenic
organs are tissues with high SR-BI expression
(21,
31). Previous in vivo studies
have established that liver, and especially the adrenals, have high rates of
selective uptake of HDL CE (4,
1012).
By simultaneously using trapped ligands for HDL apolipoproteins
(125I-TC) as well as HDL-CE ([3H]cholesteryl ether) in
the same mice, we quantitated selective HDL-CE uptake in specific tissues in
sPLA2-transgenic mice in response to the APR. Transgenic expression
of sPLA2 significantly increased the selective uptake of HDL-CE in
both liver and adrenals at baseline. Induction of the APR did not
significantly increase selective HDL-CE uptake in the liver in either
wild-type or sPLA2-transgenic mice. In contrast, induction of the
APR induced a significant increase in selective HDL-CE uptake in the adrenals
in wild-type mice and especially in sPLA2-transgenic mice. The rate
of selective HDL-CE uptake in LPS-injected sPLA2-transgenic mice
was 2.4-fold that in LPS-injected wild-type mice, indicating that
sPLA2 expression and upregulation by inflammation markedly increase
cholesterol uptake from HDL by the adrenals. The most striking metabolic
difference between sPLA2-transgenic mice and wild-type littermates
after the induction of the APR is the magnitude of selective uptake into the
adrenals. Although not specifically explored in this study, there is also the
possibility of sPLA2-mediated targeting of HDL CE to
monocytes/macrophages during the APR, because SR-BI is expressed on these
cells (36).
Expression of sPLA2 confers protection of mice against bacterial
infections, which has been demonstrated for gram-positive
(23) as well as gram-negative
(22) bacterial strains. This
has been ascribed at least in part to a direct bactericidal effect of the
enzyme (37,
38), possibly due to
degradation of bacterial surface phospholipids
(39). However, although
sPLA2 is able to kill gram-positive bacteria in vitro
(29,
38), an efficient defense
against gram-negative bacteria requires additional cofactors, such as
components of the complement system
(25,
39). Furthermore, neither
serum nor peritoneal lavage fluid from sPLA2-transgenic mice was
bactericidal against E. coli in vitro
(22), suggesting that other
properties of sPLA2 may have resulted in the increased survival of
sPLA2-transgenic mice challenged with bacteria. During acute
inflammation, there is a requirement of increased adrenal steroid hormone
synthesis, for which HDL-CE is a known source
(17,
28,
30,
41).
Our results suggest that one possible physiological role of the
sPLA2 enzyme during inflammation is to modify HDL to make HDL CE
more accessible for selective uptake into the adrenals. There are three main
families of steroid hormones synthesized by the adrenal cortex:
mineralocorticoids in the zona glomerulosa, glucocorticoids in the zona
fasciculata, and androgens in the zona reticularis
(5,
41). Both mineralocorticoids
and glucocorticoids could be helpful in the response to acute inflammation.
Although there might be species differences in the preferential usage of LDL-
or HDL-CE for steroid hormone synthesis
(36), a significantly higher
expression of SR-BI in the zona fasciculata of the adrenal cortex in mice
compared with the zona glomerulosa has been reported
(30). SR-BI-mediated uptake is
most likely the major route for the delivery of HDL-CE to the steroidogenic
pathway in the adrenals (36,
41). It is therefore probable
that, during the APR, CE from sPLA2-modified HDL is preferentially
taken up by zona fasciculata cells and used for glucocorticoid synthesis. This
extends the role of sPLA2 in the host response to bacterial
infections beyond a direct antibacterial effect and might contribute to the
significantly increased survival of sPLA2-transgenic mice in
response to a bacterial challenge.
In summary, we characterized changes in HDL catabolism during the APR in
wild-type and sPLA2-transgenic mice. Both groups of mice had
increased holoparticle catabolism by the liver and increased selective HDL-CE
uptake by the adrenals in response to LPS injection. The
sPLA2-transgenic mice demonstrated an especially robust increase in
adrenal selective HDL-CE uptake in response to induction of the APR. We
suggest that one physiological in vivo role of sPLA2 is to
significantly increase selective HDL-CE uptake by the adrenals to help meet
the requirements of increased steroid hormone synthesis during acute systemic
inflammation. This metabolic effect might be beneficial in the response to
bacteremia and other acute inflammatory insults and might contribute to
improved host survival in this setting.
 |
DISCLOSURES
|
---|
The sPLA2-transgenic mice were produced by Xenogen Biosciences
(formerly DNX Transgenic Sciences). This work was supported by National Heart,
Lung, and Blood Institute Grant HL-55323 (to D. J. Rader). D. J. Rader is an
Established Investigator of the American Heart Association and a recipient of
the Burroughs Wellcome Foundation Clinical Scientist Award in Translational
Research. U. J. F. Tietge was a recipient of a reasearch fellowship from the
Deutsche Forschungsgemeinschaft and the American Heart Association,
Pennsylvania-Delaware Affiliate. C. Maugeais was supported by a research
fellowship from the American Heart Association, Pennsylvania-Delaware
Affiliate.
Current address for U. J. F. Tietge: Dept. of Medicine, Charité
Campus Mitte, Humboldt University, Schumannstr. 20/21, D-10117 Berlin,
Germany.
 |
ACKNOWLEDGMENTS
|
---|
We are indebted to Anthony Secreto, Anna Lillethun, and Linda Morrell for
excellent technical assistance.
 |
FOOTNOTES
|
---|
Address for reprint requests and other correspondence: D. J. Rader, Univ. of
Pennsylvania Medical Center, 654 BRB II/III, 421 Curie Blvd., Philadelphia, PA
19104-6160 (E-mail:
rader{at}mail.med.upenn.edu).
Submitted 30 December 2002
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.
 |
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