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Experimental Therapeutics and
* Experimental Medicine and Nephrology, and
** Cardiovascular Biochemistry, St. Bartholomew’s and the Royal London SMD, Queen Mary and Westfield College, London EC1M 6BQ, UK; and
Institute of Pharmacology, School of Medicine, University of Messina, Messina 98123, Italy
1Correspondence: Experimental Therapeutics, St. Bartholomew’s and the Royal London SMD, Queen Mary and Westfield College, Charterhouse Square, London EC1M 6BQ, UK. E-mail: g.w.cockerill{at}mds.qmw.ac.uk
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: cytokines • chemokines • intercellular adhesion molecule 1 • interleukin 8 • multiple organ failure
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 2)
. Thus, there is a great need for new approaches to improve
therapy and the outcome of patients with HS (2)
. In
clinical practice, HS leads to a delayed vascular decompensation
(resulting in severe hypotension) and, in 
25% of patients, in the
dysfunction or failure of several organs including lung, kidney, gut,
liver, and brain (3)
. There is evidence that both ischemia
(due to reduced blood and oxygen supply during hemorrhage) and
reperfusion (during resuscitation) play an important role in the
pathophysiology of the multiple organ dysfunction syndrome (MODS) in HS
(4)
.
In both experimental and clinical studies, local or generalized
ischemia (followed by reperfusion) resulted in increased vascular
permeability leading to protein leakage, formation of
noncardiogenic interstitial edema, and the accumulation of
neutrophils in all organs (5
6
7
8)
. The infiltration of
neutrophils into tissues is determined largely by interplay between
cytokines, chemokines, and adhesion molecules (9)
. Several
lines of evidence suggest that adhesion blockade may be a useful
therapeutic approach (10
, 11)
. Although the precise
factors leading to MODS have not been identified (12)
, the
rapid increase of cytokines and adhesion molecules very early after
trauma and hemorrhage and the rapid decrease in interleukin 10 (IL-10)
in patients who develop MODS (13)
suggest a loss of
anti-inflammatory potency.
Plasma high density lipoproteins (HDLs) are a family of mostly
spheroidal particles of density 1.063–1.21 g/ml. As they are smaller
than other lipoproteins, they penetrate between the endothelial cells
more readily, producing relatively high concentrations in tissue fluids
(14)
. The major apolipoprotein (apo) of almost all plasma
HDLs is apo A-I, which in association with phospholipids and
cholesterol encloses a core of cholesteryl esters. Nascent (i.e., newly
synthesized) HDLs secreted by liver and intestine contain no
cholesteryl esters, and are discoidal. A negative association of plasma
HDL concentration with coronary artery disease has been documented from
epidemiologic studies (15
16
17)
. Experiments in animals
have demonstrated that HDLs have direct anti-atherogenic activity
(18
19
20
21)
.
We have shown that HDLs are able to inhibit cytokine-induced expression
of adhesion molecules and that their anti-inflammatory properties are
also observed in animal models (22
23
24)
. In this study, we
investigated the hypothesis that systemic administration of HDLs will
exert beneficial effects in animal models of HS. We have examined the
effects of native high density lipoproteins (nHDLs) and reconstituted
high density lipoproteins (recHDLs) on the organ injury and failure
caused by severe hemorrhage and resuscitation in rats, particularly the
effects of nHDLs and recHDLs on renal dysfunction and liver,
pancreatic, intestinal, and lung injury associated with HS. To gain
better insight into the mechanism of the beneficial effects of HDLs
observed in this model, we also investigated their effects on
1) adhesion and transmigration of polymorphonuclear
leukocytes (PMNs) and 2) cytokine-induced synthesis of IL-8
in human umbilical vein endothelial cells (HUVECs) in vitro and the rat
IL8 homologue macrophage inflammatory protein 2 (MIP-2) in rats in
vivo. We also investigated the effects of HDLs on the expression of
ICAM-1 and P-selectin in the kidney and intestine of rats subjected to
shock.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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METHODS
Isolation of nHDLs
Blood from healthy humans, aged 25–40 years, was collected in
VacutainersTM containing K2EDTA as the
anticoagulant (Becton Dickinson, Franklin Lakes, NJ). After separating
plasma by centrifugation at 2000 rpm for 20 min at 4°C, nHDLs were
isolated in the 1.07–1.21 g/ml density range by sequential preparative
ultracentrifugation (25)
.
Reconstituted HDLs
The discoidal recHDLs were provided by the Central Laboratory,
Swiss Red Cross, Bern. The particles, containing human apo A-I as the
sole protein and soybean phosphatidylcholine as the sole phospholipid
were prepared using cholate dialysis (26)
. Their
physicochemical properties have been described in detail
(27)
.
Cell culture
HUVECs, isolated as described previously (28)
, were
grown on gelatin-coated tissue culture flasks (Costar, High Wycombe,
Bucks, UK) in Medium 199 with Earle’s salts supplemented with 20%
fetal calf serum (FCS), 20 mM HEPES, 2 mM glutamine, 1 mM sodium
pyruvate, nonessential amino acids, penicillin, streptomycin, 50
µg/ml endothelial cell growth supplement (Sigma, Dorset, UK.), and 50
µg/ml heparin.
Neutrophil isolation and labeling
Neutrophils were isolated from the whole blood of normal healthy
humans by Ficoll/Hypaque (Pharmacia, Piscataway, NJ) gradient
centrifugation and dextran sedimentation with hypotonic lysis of
contaminating erythrocytes. Radiolabeled neutrophils were prepared
according to the method of Gamble et al. (29)
.
Neutrophil adhesion assay
HUVECs were plated at 105
cells/cm2 in Medium 199 containing 20% FCS in
microtiter wells (Nunc, Naperville, IL) and grown to confluence. Some
cells were treated with either recHDLs or nHDLs (1 mg/ml apo A-I) for
4 h before the addition of PBS or tumor necrosis factor 
(TNF-) (10 U/ml). Cells were incubated an additional 4 h before
the addition of neutrophils, all monolayers were washed once with
Medium 199 containing 5% FCS; 100 µl of the foregoing medium
containing 5 x 105 51Cr-labeled neutrophils
was added to each well. The plates were then agitated to evenly
disperse the neutrophils. Cells were incubated at 37°C in 5%
CO2 for 30 min, after which the nonadherent
neutrophils were aspirated and each well was washed three times with
Medium 199 containing 5% FCS. The cell monolayer and adherent
51Cr-labeled neutrophils were then lysed for at
least 2 h with 1 M ammonium hydroxide. Adherence was evaluated as the percentage of total 51Cr
cpm added: i.e., percentage adherence =
(51Cr cpm in lysate/total
51Cr cpm added) x 100.
Neutrophil transmigration assay
The method used was described previously
(30)
. Monolayers grown on TranswellsTM (6.5 mm diameter, 8
µm pore size) were incubated with either PBS, recHDLs, or nHDLs (1
mg/ml apo A-I) for 4 h before activation by the addition of
TNF- (10 U/ml) an additional 4 h. All wells were then washed.
Chromium-labeled neutrophils (106/well) were
added and the TranswellTM was placed in a fresh 24-well culture tray
containing HUVEC medium with 5%FCS. Anti-IL-8 antibody (1/300
dilution) was added to the lower compartment of some groups. After
1 h incubation with 5% CO2 at 37°C, the
amount of transmigration was calculated by gamma counting the
cells in the lower chamber. Extent of migration was calculated as
a percentage of the total cells added.
IL-8 ELISA measurement
Endothelial cells were plated onto gelatin-coated 96-well
microtiter plates at 1 x 105
cells/cm2 in Medium 199 (containing 20% FCS and
the normal growth supplements) and grown to confluence. RecHDLs were
added to some wells (1 mg/ml apo A-I) and the cultures were incubated
an additional 4 h. TNF- (10 U/ml) was added to some cultures
pretreated with recHDLs and to some cultures grown without any
addition. Four hours after the addition of cytokine, the medium was
gently aspirated from all cultures and replaced with fresh medium. IL-8
was assayed using an ELISA (R&D Systems, Cambridge, UK) in supernatants
collected 1 h after changing the medium. A standard dose response
curve of IL-8 was measured both with and without the addition of
recHDLs at 1 mg/ml apo A-I in order to observe any effect of the
lipoproteins on the ELISA assay.
Rat model of HS
All experiments were approved by the Institutional Animal
Research Committee and performed in adherence to Guide for the Care and
Use of Laboratory Animals published by the U.S. National Institutes of
Health (NIH publication No 85–23, revised 1996).
Surgical procedure
These studies were carried out, as described previously
(31)
, on male Wistar rats (Tuck, Rayleigh, Essex, UK) of
250–320 g receiving a standard chow and water ad libitum. All animals
were anesthetized with thiopentone (120 mg/kg intraperitoneal) and
anesthesia was maintained by injections of thiopentone as required. The
trachea was cannulated to facilitate respiration and rectal temperature
was maintained at 37°C with a homeothermic blanket. The right femoral
artery was catheterized and connected to a pressure transducer
(Senso-Nor 840, Senso-Nor, Horten, Norway) to measure phasic and mean
arterial blood pressure (MAP) and heart rate, which were displayed on a
data acquisition system (MacLab 8e, ADI Instruments, Hastings, UK)
installed in an Apple Macintosh computer. The right carotid artery was
cannulated to bleed the animals and the jugular vein was cannulated for
the administration of drugs. The bladder was cannulated to facilitate
urine flow and to prevent the development of postrenal failure. Upon
completion of the surgical procedure, cardiovascular parameters were
allowed to stabilize for 15 min; then blood was withdrawn from the
carotid artery catheter in order to achieve a fall in MAP to 50 mmHg
within 10 min. MAP was maintained at 50 mmHg for 90 min by withdrawal
(during the compensation period) or reinjection of blood. In none
of the experiments did the amount of shed blood reinjected during the
90 min period of hemorrhage exceed 10% of the total amount withdrawn.
At 90 min after initiation of hemorrhage, the remaining shed blood was
reinjected into the animal. At the same time an intravenous (i.v.)
infusion of isotonic saline (1.5 ml/kg/h) was started as fluid
replacement and maintained throughout the experiment.
Experimental protocol
The following experimental groups were studied:
1. Sham-control group: Rats were subjected to the surgical procedure without causing a hemorrhage and treated with vehicle for HDLs (saline, 1 ml/kg i.v. bolus, followed by an infusion of 1.5 ml/kg/h i.v.; n=9).
2. Sham-recHDL control group: Rats were subjected to the same surgical procedure without causing a hemorrhage, but received recHDLs (80 mg apo A-I/kg i.v. bolus, followed by an infusion of 1.5 ml/kg/h i.v.; n=5).
3. Hemorrhage-control group: One minute before resuscitation with the shed blood, the rats were treated with vehicle (saline, 1 ml/kg i.v. bolus, followed by an infusion of 1.5 ml/kg/h i.v.; n=7).
4. Hemorrhage-recHDL group: At 1 min before resuscitation, animals received a bolus injection of recHDLs (80 mg apo A-I/kg i.v.bolus, followed by 1.5 ml/kg/h i.v.; n=10).
5. Hemorrhage-nHDL group: At 1 min before resuscitation, animals received a bolus injection of nHDLs (80 mg apo A-I/kg i.v.bolus, followed by 1.5 ml/kg/h i.v.; n=10).
Quantification of organ function and injury
Blood samples (0.5 ml) were taken at baseline (t=0) and at
4 h after resuscitation (end of the experiment) from a right
carotid artery catheter into serum gel S/1.8 tubes (Sarstedt, Germany).
The samples were centrifuged (1610 g for 3 min) to separate
serum. All sera samples were analyzed within 24 h by a specialized
service laboratory (Vetlab Services, Sussex, UK). The following
biochemical markers of tissue dysfunction were measured: 1)
liver injury was assessed by assaying alanine aminotransferase (ALT, a
specific marker for hepatic parenchymal injury) and aspartate
aminotransferase (AST, a nonspecific marker for hepatic injury)
(32
, 33)
; 2) renal dysfunction was assessed by
measuring the rises in plasma levels of creatinine (an indicator of
reduced glomerular filtration rate, and hence of renal failure) and
urea (an indicator of impaired excretory function of the kidney and/or
increased protein catabolism) (34)
; 3) serum
total lipase activity was determined as an indicator of pancreatic
injury; 4) total creatine kinase activity was measured as an
indicator of neural and/or muscular injury.
Light microscopy
Organ (lung, kidney, and small intestine) biopsies were taken at
the end of the experiment and fixed for 1 wk in buffered formaldehyde
solution (10% in PBS) at ambient temperature, dehydrated by graded
ethanol, and embedded in ParaplastTM (Sherwood Medical, Mahwah, NJ).
Sections (7 µm thick) were deparaffinize with xylene, stained using
either Van Gieson’s Trichrome or Fuchsin, and examined using light
microscopy (Dialux 22, Leitz).
Immunohistochemistry
The expression of ICAM-1 and P-selectin was evaluated by
immunohistochemistry in biopsies from kidney and small intestines. At
the end of the experiment, the organs were fixed as described above.
The sections were permeabilized with 0.1% Triton X-100 in PBS for 20
min. Nonspecific adsorption was minimized by incubating the sections in
2% normal goat serum in PBS for 20 min. Endogenous biotin or avidin
binding sites were blocked by sequential incubation for 15 min with
avidin or biotin. The sections were then incubated overnight with
1:1000 dilution of primary antibody or control solution. Controls
included buffer alone or nonspecific purified rabbit IgG. Specific
labeling was detected using a biotin-conjugated goat anti-rabbit IgG
and avidin-biotin peroxidase complex formation. All immunohistological
evaluations were carried out by an observer who was blinded as to the
particular treatment of each specimen.
Determination of myeloperoxidase activity
Myeloperoxidase (MPO) activity, an indicator of PMN accumulation
was determined as described previously (35)
. Samples of
lung and kidney were obtained and weighed. Each piece of tissue was
homogenized in a solution containing 0.5%
hexa-decyl-trimethyl-ammonium bromide dissolved in 10 mM potassium
phosphate buffer (pH 7.0) and centrifuged for 30 min at 20,000
g at 4°C. An aliquot of the supernatant was then allowed
to react with a solution of tetra-methyl-benzidine (1.6 mM) and 0.1 mM
H2O2. The rate of change in
absorbance was measured spectrophotometrically at 650 nm; 1 mU of MPO
activity was defined as the quantity of enzyme degrading 1 µmol of
peroxidase per min at 37°C and was expressed in mU per mg of wet
tissue.
Determination of malondialdehyde
Malondialdehye (MDA) levels in the lung and kidney were
determined as an indicator of lipid peroxidation. Tissues were
homogenized in 1.15% KCl solution. An aliquot (100 µl) of the
homogenate was added to a reaction mixture containing 200 µl 8.1%
SDS, 1500 µl 20% acetic acid (pH 3.5), 1500 µl 0.8%
thiobarbituric acid, and 700 µl distilled water. Samples were then
boiled for 1 h at 95°C and centrifuged 3000 g for 10
min. The absorbance of the supernatant was measured
spectrophotometrically at 650 nm.
RNase protection assay
Renal biopsies were collected from each animal 4 h after
resuscitation. Tissues were stored in liquid nitrogen until required
for total RNA extraction. MIP-2 mRNA levels relative to ß-actin were
determined by RNase protection (36)
. Sp6 RNA polymerase
transcription templates for the preparation of
32P-labeled RNA probes used in the RNase
protection assay contained a 282 bp fragment of rat MIP2 (a gift from
Joseph Paulaskis) and a 354 bp fragment of rat ß-actin.
Statistical evaluation
All data are presented as means ± SE of
n observations, where n is the number of animals,
blood samples, or culture assays. For repeated measurements (e.g.,
hemodynamics), a 2-factorial analysis of variance (ANOVA) was
performed. Data without repeated measurements (multiple organ
injury/failure) were analyzed by a 1-factorial ANOVA, followed by a
Dunnett’s test for multiple comparisons. A P value of <0.05 was considered to be statistically significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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HDLs are anti-inflammatory in vitro and inhibit the adhesion and
transmigration of neutrophils in an IL-8-dependent manner
The ability of HDLs to inhibit the adhesion and
transmigration of neutrophils was measured using standard in vitro
models. Treatment of confluent endothelial cells with nHDLs or recHDLs
(1 mg apo A-I/ml) inhibited neutrophil adhesion by 72.4 ± 6.3%
(n=6, P<0.05) and 68 ± 5.9%
(n=6, P<0.05), respectively (Fig. 1A
). The extent of inhibition of neutrophil transmigration by
HDLs (Fig. 1B
), where treatment of confluent endothelial
cells grown on TranswellsTM with either nHDLs or recHDLs (1 mg apo
A-I/ml), inhibited TNF--induced transmigration by 48.7 ± 6.7%
(n=8, P<0.05) and 46.2 ± 8.4%
(n=8, P<0.05), respectively. The ability of HDLs
to inhibit transmigration of neutrophils was not significantly
different from that observed using blocking anti-IL-8 antibodies:
44.7 ± 8.4% (n=8, P<0.05) (Fig. 1B
). The addition of blocking anti-IL-8 antibodies after
treatment with either nHDLs or recHDLs (1 mg apo A-I/ml) did not
further inhibit the extent of transmigration, suggesting that HDLs
inhibit the IL-8-dependent component of neutrophil transmigration. We
investigated the ability of HDLs to affect the cytokine-induced
expression of IL-8 in endothelial cells. Treatment of the endothelial
cells with either nHDLs or recHDLs (1 mg apo A-I/ml) ablated the
TNF--induced production of the chemokine (Fig. 1C
).
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HDLs had no effect on the hemodynamic responses to
hemorrhage and resuscitation in the rat model
Baseline values of MAP in all groups of animals ranged
from 127 ± 3 to 141 ± 7 mm Hg and were not significantly
different between groups (Fig. 2
). In sham-operated rats (no hemorrhage), neither nHDLs nor recHDLs had
a significant effect on MAP (Fig. 2)
. In rats resuscitated with shed
blood, there was an immediate increase in blood pressure from 50 mmHg to 113 ± 4 mmHg. Thereafter, there was a progressive
slow decline in MAP to 65 mmHg by the end of the experiment (Fig. 2)
. Administration of nHDLs or recHDLs at 1 min before
resuscitation had no significant effect on the delayed fall in MAP
associated with hemorrhage. Baseline values of heart rate in all groups
of animals ranged from 364 ± 13 to 416 ± 10 beats per min
(bpm) and were not significantly different between groups (Table 1). In control animals, administration of nHDLs or recHDLs did not result
in any significant alteration in heart rate. Hemorrhagic shock did not
cause a significant alteration in heart rate (Table 1)
.
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Effects of nHDLs or recHDLs on the multiple organ
dysfunction syndrome caused by hemorrhage and resuscitation in the rat
HDLs prevent the elevation of serum markers of tissue
dysfunction
Effects on renal dysfunction
In sham-operated rats, administration of saline, nHDLs,
or recHDLs did not result in any significant alteration in the serum
levels of urea (Fig. 3A
) or creatinine (Fig. 3B
). Compared with
sham-operated rats, hemorrhage/resuscitation resulted in significant
rises in the serum levels of urea and creatinine. Treatment of rats
subjected to hemorrhage with nHDLs or recHDLs before resuscitation
abolished the increase in urea and creatinine caused by hemorrhage
(Fig. 3A
, B
).
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Effects on liver injury
In sham-operated rats, administration of saline, nHDLs,
or recHDLs did not result in any significant alteration in the serum
activities of AST (Fig. 3C
) or ALT (Fig. 3D
).
Compared with sham-operated rats, hemorrhage/resuscitation resulted in
a significant rise in the serum activities of AST. Treatment of rats
subjected to hemorrhage with nHDLs or recHDLs before resuscitation
abolished these enzyme changes (Fig. 3C
, D
).
Effects on pancreatic injury
Administration of saline, nHDLs, or recHDLs in sham-operated
rats did not result in any significant alteration in serum total lipase
activity (Fig. 3E
). Compared with the sham-operated
rats, hemorrhage/resuscitation resulted in a significant rise in serum
total lipase activity. Treatment of rats subjected to hemorrhage with
nHDLs or recHDLs before resuscitation abolished this effect (Fig. 3E
).
Effects on neural and/or muscular injury
The administration of administration of saline, nHDLs, or recHDLs
In sham-operated rats did not result in any significant alteration in
the plasma activity of total creatine kinase (CK) activity (Fig. 3F
). Compared with the sham-operated rats,
hemorrhage/resuscitation resulted in a significant rise in serum total
CK activity, demonstrating the development of brain and/or muscular
injury. Treatment of rats subjected to hemorrhage with recHDLs
before resuscitation ablated the rise in total CK activity (Fig. 3F
). No significant differences were observed when
rats were given nHDLs before resuscitation. To measure any interference
by exogenous HDLs on the assays used to measure markers of MODS, we
added recHDLs (apo A-I 1 mg/ml) to sera samples from group 1
(sham-vehicle) and group 3 (HS-vehicle). No significant differences
were observed in the measured levels of these markers in the presence
and absence of added recHDLs.
Organ dysfunction as measured by the degree of disruption of tissue
architecture was reduced by treatment with HDLs
Compared with organs obtained from sham-operated rats that
had not been subjected to hemorrhage and resuscitation (data not
shown), the lung (Fig. 4
, top panels), small intestine (middle panels), and kidney (bottom
panels) subjected to hemorrhage and resuscitation (Fig. 4A
) showed edema with loss of normal tissue structure.
In contrast, organs from animals that had received nHDLs (Fig. 4B
), or recHDLs (Fig. 4C
) before
resuscitation showed no significant change in morphology, and were not
significantly different from the sham-operated rats (not shown).
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There is less cellular infiltration in the photomicrographs of organs from animals that had received HDLs. As this infiltration of leukocytes is dependent on the expression of adhesion molecules in these tissues, we examined the effect of HDLs on the expression of ICAM-1 and P-selectin.
Expression of P-selectin and ICAM-1 after hemorrhage and
resuscitation was reduced by HDLs
When compared with organs obtained from sham-operated rats (not
shown), the small intestine (top panel) and kidneys (bottom panel) of
rats subjected to hemorrhage and resuscitation (Fig. 5A
) showed staining for ICAM-1 on both the epithelium and
endothelium. In contrast, the degree of staining for ICAM-1 was visibly
reduced in rats treated with nHDLs (Fig. 5B
) or recHDLs
(Fig. 5C
) and not visibly different from the sections from
the sham-operated animals (not shown).
|
Compared with organs obtained from sham-operated rats (not shown), the
small intestine (bottom panel) and kidneys (top panel) of rats
subjected to hemorrhage and resuscitation (Fig. 6A
) also showed staining for P-selectin on the endothelium. In
contrast, the degree of staining for P-selectin was visibly reduced in
rats that had been treated with nHDLs (Fig. 6B
) or recHDLs
(Fig. 6C
). We have no evidence that staining for P-selectin
is due to the presence of the protein in platelets (e.g., located in
microthrombus).
|
HDLs reduced neutrophil infiltration into lungs and kidneys after
hemorrhage and resuscitation
The ability of HDLs to inhibit the expression of adhesion
molecules in this model is strongly supported by the histology data. We
further investigated the ability of HDLs to inhibit neutrophil
infiltration by measuring MPO levels the lung (Fig. 7A
) and kidney (Fig. 7B
). Compared with tissues
obtained from sham-operated rats, rats subjected to hemorrhage and
resuscitation (solid bars) showed an increase in tissue MPO activity
that was reduced in rats treated with either nHDLs or recHDLs before
resuscitation with shed blood.
|
HDLs reduced malondialdehyde levels in lungs and kidneys after
hemorrhage and resuscitation
Since HDLs have been shown to have antioxidant properties, we
investigated the ability of HDLs to influence MDA levels the lung
(Fig. 8A
) and kidney (Fig. 8B
). Compared with
tissues obtained from sham-operated rats, rats subjected to hemorrhage
and resuscitation (solid bars) showed a marked increase in tissue MDA
activity that was reduced in rats treated with either nHDLs or recHDLs
before resuscitation.
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The HDL-mediated reduction of neutrophil infiltration in the kidney
is accompanied by a significant reduction in the mRNA levels of MIP-2
In Fig. 1
we show that HDLS are able to inhibit the neutrophil
chemokine IL-8. To examine whether HDL given during resuscitation was
able to reduce the expression of the rat chemokine, we compared the
mRNA concentration of MIP-2, the rat IL-8 homologue, in kidney biopsies
collected 4 h postresuscitation between animals subjected to
hemorrhage and resuscitation given either recHDL upon resuscitation
(HS/recHDL) or carrier alone upon resuscitation (HS/PBS). The level of
MIP-2 relative to ß-actin is shown in six animals from each group in
Fig. 9
. In animals given recHDL during resuscitation, there is a marked
reduction in the relative levels of MIP-2 vs. those animals that
received carrier alone. This result is consistent with those
observations made in human cells in vitro.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 38
, 39)
,
we investigated the ability of HDLs to alleviate organ
injury/dysfunction in a rat model of severe HS. Administration of HDLs
did not alter the hemodynamics in this model, consistent with studies
of a porcine model of HS (40)
. However, HDLs did attenuate
the organ injury/dysfunction induced in our model of HS. We postulate
that elevation of HDLs provides the anti-inflammatory potency that is
reduced during the development of MODS. Several aspects of the effects
of HDLs on endothelium and leukocytes could have contributed to their
protective effects in our model.
HDLs might have had a direct effect on the expression of adhesion
molecules. The demonstration that HS leads to the expression of
adhesion molecules (41)
has prompted several groups to
examine the beneficial effects of specific inhibitors, such as soluble
P-selectin ligand (sPSGL-1) and blocking antibodies to CD18, in a model
of ischemia and reperfusion injury (42
, 43)
. In those
studies, inhibition of adhesion molecules perturbed the interaction
between endothelium and leukocytes and increased survival rate. In a
similar model of severe HS in rats, both dextran and the xanthine
oxidase inhibitor pentoxifylline reduced the level of expression of
P-selectin (44)
. In our study, a decrease in the level of
expression of ICAM-1 and P-selectin further supported the
anti-inflammatory role of HDLs and could partly explain their effects
in this model.
The protective effect of HDLs could be mediated in part through effects
on chemotactic factors important in leukocyte transmigration. In
addition to the demonstration by Fogelman and colleagues that HDLs
could reduce the synthesis of monocyte chemotactic factor 1
(45)
, we show the ability of HDLs to partially inhibit
neutrophil transmigration through inhibition of the C-X-C chemokine
IL-8. The rat homologue of this molecule, MIP-2, was originally
identified in LPS-stimulated mouse macrophages (46)
and
characterized as a potent chemokine for neutrophils in vitro
(46)
and in vivo (47
48
49)
. MIP-2 has been
shown to be produced by mesangial cells and associated with neutrophil
influx in glomerular nephritis (50)
and ischemia
reperfusion injury (51)
. Regulation of levels of MIP-2 is
shown to be a combination of enhancement of transcription and
post-transcriptional stabilization, responsible for the increase in
MIP-2 mRNA induced by oxidative stress (52)
. Our results
show that elevation of HDL during resuscitation significantly perturbs
the level of MIP-2 normally induced in the kidney during hemorrhage and
resuscitation.
As the pathophysiologic processes involved in MODS are known to be
influenced greatly by reactive oxygen species, the antioxidant
properties of HDLs may play a part in their protective effect
(53
, 54)
. The antioxidant properties of HDLs are related
in part to the presence of paraoxonase in some particles
(55)
and partly to an independent property of apo A-I
(56)
. The present results are unlikely to have been due to
an effect of paraoxonase, as the recHDLs, unlike the nHDLs, contained
apo A-I as the sole protein component. However, the antioxidant
activity of apo A-I might have accounted for part of the effect of HDLs
in the present model. This possibility was supported by the ability of
HDLs to reduce lipid peroxidation in the lungs and kidneys of rats
subjected to HS.
HDLs have been shown to bind endotoxins in vitro (57)
and
to prevent the pathophysiologic sequelae of endotoxin infusion in vivo
(58)
. Endotoxemia is a known consequence of ischemia of
the alimentary tract (59)
. Therefore, we must also
consider the possibility that the ability of HDLs to bind endotoxins
might have contributed to the protective effect of the lipoprotein in
our model. However, Magnotti and colleagues have recently reported that
in a similar model of severe HS in rats, they were unable to detect
either bacteria or endotoxin in mesenteric lymph or portal vein plasma
(60)
. This is consistent with other work showing that in
patients with severe trauma, bacteria and endotoxins were not
detectable in plasma (61
, 62)
. However, a partial effect
cannot be ruled out in our experiments.
In conclusion, we show that the administration of HDLs before resuscitation ablates the infiltration and activation of neutrophils and prevents organ injury and dysfunction induced by HS. Further studies are required to understand the mechanism of action of HDLs in this regard. The extent and breadth of the benefits of HDLs in this model suggest that recHDLs might be of therapeutic value in the treatment of severe trauma.
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ACKNOWLEDGMENTS |
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Received for publication May 16, 2000.
Revision received April 30, 2001.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) and recHDLs (
) on
neutrophil adhesion (A), neutrophil transmigration
(B), and TNF-
