Goins,. B., Phillips,. W.T.. (1991). In vivo biodistnibution of a radiolabeled blood substitute: 99mTclabeled liposome-encapsulated hemoglobin in an anesthetized.
Liposome-encapsulated factor
release
Macrophages response
of Surgery,
that
alveolar
macrophages
V. Maier,
University
contribute characterizes
of
to
Washington
Lynne and
the systemic the sepsis syn-
shock
cytokine
necrosis
by a
. sepsis
. blood
Timothy
Affairs
H. Pohlman,
Medical
Center,
John
G. Williams,
Seattle
Liposome clearance from the circulation is primarily via fixed tissue macnophages in the liver and spleen, with accumulations in the lung being dependent on the size of the liposome [5, 6]. When stimulated, these cells are a major source of inflammatory mediators that have been implicated in the diffuse organ injury seen during multiple organ failure
dependent of the period of exposure to the LEH. Despite inhibition of TNF expression, Northern blot analysis of total cellular RNA from LPS-stimulated macrophages revealed accumulations of TNF-specific transcripts in cells treated with or without LEH. Thus the mechanism of LEH inhibition of TNF expression appears to involve a posttranscriptional event. Although these results suggest a potential advantage of resuscitation with LEH when sepsis complicates hemorrhagic shock, immunomodulation in vivo remains to be defined. J. Lcukoc. Biol. 52: 679-686; 1992. Words:
Wilson,
Veterans
drome through the production of inflammatory cytokines such as tumor necrosis factor (TNF). Liposomeencapsulated hemoglobin (LEH), a potential red cell substitute, is cleared by fixed tissue macrophages. In these studies, in vitro incubation of alveolar macrophages with stored LEH was shown to inhibit the expression of TNF induced by endotoxin (lipopolysaccharide, LPS) stimulation. This effect was dependent on LEH dose but in-
Key
tumor
mechanism1
Lorrie A. Langdale, Ronald and Charles L. Rice
Abstract: inflammatory
inhibits
from rabbit
posttranscriptional Department
hemoglobin
substitute
[ 7]. Since macnophages play a central role in LEH clearance as well as the cellular response to injury, reticuloendothelial system modulation by LEH in the settings of major trauma resuscitations, complex surgeries, or infection might prove to be clinically significant. Although it is unlikely that phagocytosis of liposomes would directly induce macrophage activation, there is precedent for cellular “priming” of macnophages by phagocytosed mediators [8-10]. Endotoxin stimulation of LEH-pnimed macrophages might produce an exaggerated tumor necrosis factor (TNF) response, inducing the equivalent of endotoxic shock in the presence of what would otherwise be a clinically insignificant gram-negative infection. Altennatively, macrophage ingestion of LEH may inhibit the effects of lipopolysacchanide (LPS) via down-regulation of the macrophage response to stimulation. The present study investigated the effects of LEH on macrophage function, as defined by transcription and expression of TNF, to
by examining
endotoxin
the
response of alveolar in vitro exposure
stimulation
after
macnophages to LEH.
INTRODUCTION Criteria for a clinically useful temporary red cell substitute demand that it remain in circulation long enough to sustain adequate oxygen and carbon dioxide transport until sufficient red cell mass is available, either by traditional transfusion or normal hematopoietic production. Use of such a product in resuscitation after hemorrhagic shock or trauma would obviate the need for type and cross matching and have the additional advantage of an extended shelf life. An alternative oxygen carrier must also be removed in a manner that is not toxic to organ or cellular function. Liposome-encapsulated hemoglobin (LEH) is currently being evaluated as a potential red cell substitute. Encapsulation of stroma-free hemoglobin in unilamellar lipid vesicles has been shown to sustain the circulating half-life beyond that of unencapsulated hemoglobin products, while retaining 02 and CO2 transport capabilities [1]. Important safety issues, however, including acute and delayed effects on major organ system and cellular function, as well as the impact of prolonged storage, have not been fully investigated. Smallvolume infusions in laboratory animals and clinical trials using liposomes as drug carriers have found no shortor longterm hematological or hepatic effects [2-4]. The effects of large-volume infusions of lipid vesicles encapsulating a complex protein such as hemoglobin are unknown.
MATERIALS
AND METHODS
Materials LEH was obtained through the Naval Research Laboratories (Bethesda, MD). It was made in accordance with the method described by Farmer ct al. [1], encapsulating bovine stromafree hemoglobin in lipid vesicles composed of a 10:9:0.9:0.1 ratio of soy phosphatidylcholine, cholesterol, dimynistoylphosphatidylglycerol, and a-tocopherol. In brief, the lipid components of the membrane were mixed in organic phase and dried into a film. Concentrated bovine stroma-frec hemoglobin was added and the suspension extruded through
Abbreviations:
mm; m,
BAL,
LEH,
bronchoalveolar
lavage;
liposome-encapsulated
macrophage;
BSA,
hemoglobin;
TNF,
tumor
necrosis
bovine
LPS,
factor;
serum
albu-
lipopolysaccharide;
VLDL,
very
low
density
lipoprotein. Reprint
requests:
Affairs
Medical
Seattle,
WA
Received This Shock,
Journal
A.
Langdale,
(Bldg.
Department
1, Rm
314),
1660
of South
Surgery,
Veteran
Columbian
Way,
98108. April
work
Lorrie Center
was
Vienna,
of Leukocyte
14,
1992;
presented Austria,
accepted in
June
Biology
July part
4,
at
1991
Volume
30, the
1992. International
(Circ.
Shock
52,
Conference 34(1):
December
96,
1992
on
1991)
679
A.
a Microfluidizen to produce predominantly unilamellar LEH approximately 200 nm in diameter [11]. The liposomes were stabilized in phosphate buffer and refrigerated in sterile vials for the duration of storage. Two batch preparations of similarly formulated LEH were tested. One batch (LEH 1) had been stored for 6 months prior to evaluation, the second (LEH 2) for 18 months. Characteristics of the solutions are given in Table 1. Phenol-extracted Escherichia coli 0111:B4 LPS (Sigma Chemical Co., St. Louis, MO) was suspended in pynogenfree saline, sonicated, aliquoted, and stored at -80#{176}C. Samples were diluted with RPMI 1640 medium (Gibco, Grand Island, NY) to achieve final experimental concentrations of 1, 10, 100, and 1000 ng/ml.
Cell preparation
and experimental
1200 I 000 Cl)
.-
C U-
z
400
I200.
design 0.0
The with
ease with which minimal perturbation
alveolar macnophagcs ofcellular function
0.5
are harvested made this cell
tO
Ratio
population ideally suited to in vitro analysis of the effects of LEH on macrophage (m) function. Cells were obtained from 1.5-2.5-kg New Zealand White, specific pathogen-free rabbits by bronchoalveolan lavage (BAL). After euthanasia with an overdose of intravenous phenobarbital, the trachea was isolated and cannulated under sterile conditions. Lungs
1.5
2.0
34
of Anti TNF to TNF
B. 30000
were gently lavaged six times with 50 ml aliquots of 4#{176}C sterile saline. Lavage solutions were centrifuged at l000g for 10 mm at 4#{176}C,the supernatant was discarded, and the cells were resuspended in RPMT 1640 containing 50 tg/ml gentamicin. Macrophages were immediately plated into 12-well tissue culture plates at 5 x 10 cells/ml/well and allowed to adhere for a minimum of 30 mm. Cells were shown to be >95% ms by csterase staining, phagocytosis oflatex beads, and Giemsa staining for differential count. The cells were >90% viable by trypan blue vital dye exclusion, and prepanations with >5% neutrophils were considered contaminated and discarded. Plated macnophages were incubated with 100 jl of LEH at 37#{176}C, 5% CO2 for 3 h. A volume-for-volume exchange ( medium for LEH on medium for medium) to maintain a well volume of 1 ml was performed in duplicate for each cxpenimental condition and control. After the appropriate incubation period, each well was washed three times with warm RPMT 1640 prior to the addition of 1 ml of RPMI 1640. Final working concentrations of 1, 10, 100, and 1000 ng/ml LPS were added to appropriate wells in 10 il aliquots, and the plates were incubated for 18-21 h to ensure maximal LPS stimulation. Supernatant to be analyzed for TNF was removed from each well and combined duplicates were centrifuged at 1000 rpm for 10 mm. To enhance TNF stability, 1 ml of the supernatant was mixed with bovine serum albumin (BSA) to a
TABLE
I
.
Characterization
of
Liposome (nm)
size
Liposome-Encapsulated
15.6 13.3
34 20
LEH 2 Original 18 months
230 248
16.2 33.2
47 50
680
10000
z I-
0
8
12
Time Fig. for
1. TNF TNF
was
TNF-specific
and
tion
at 4 h and was
observed
cytolysis by
antibody
LPS-stimulated
mences
L929
confirmed
ize the cytokine by
the
was
at a ratio continues the
(A) the
in varying
of 1:34.
alveolar over
added
(B)
L929
24
of the
using
dilutions
1200 and
measurement in
to accumulate.
No
h of
Specificity
assay
macrophages 24
20
(hrs)
assay.
performing
16
the
U
found
diminution
assay
of TNF.
A
to neutral-
ofTNF
absence
L929
of
produced serum
ofTNF
cornproduc-
stimulation.
final 1% BSA dilution and stored at -80#{176}C for later TNF analysis. Six complete experiments were performed with samples assayed in duplicate (n = 6 animals; cells from each animal subjected to each control, experimental condition, and LPS dose).
TNF assay 190 200
“Similarly
U.
He mocrit (% LEH)
(%)
LEH I Original 6 months
prolonged expected.
C
Hemoglobin”
Methemoglobin
20000
Cl)
formulated
storage.
Journal
LEH
showed
minimal
Methemoglobin
concentration
of Leukocyte
Biology
changes
in liposome
increased
Volume
size
over
52,
time,
December
TNF was measured using the biologic cytotoxicity Flick and Gifford [12]. Transformed mouse L929 (NCTC clone L929, American Type Culture
assay fibroblasts Collection,
of
Rockville, MD) were pretneated with actinomycin D (Sigma Chemical Co., St. Louis, MO) at 2 tg/ml for 15 mm in RPMI 1640 supplemented with 5% horse serum (Hyclonc Laboratories, Logan, UT) and added to the serial dilutions of the conditioned medium at 5 x 10’” cells/well. After 24 h
with
as
1992
of incubation stained monolayers ethanol of TNF
at 37#{176}Cwith
5%
C02,
the
cells
were
fixed
and
with
0.1% crystal violet in 20% methanol. Dried were solubilized in 0.1 M sodium citrate in 50% and the absorbance was read at 550 nm. One unit activity was defined as the activity that produced
50% cytolysis of the L929 monolayer. The data were analyzed by linear regression to determine the TNF dilution point at which 50% cytolysis occurred. The L929 assay was validated for TNF specificity using goat polyclonal antihuman recombinant TNF, provided by Dr. John Mathison, of the Scripps Clinical and Research Foundation, La Jolla, CA. Immunization of goats with human recombinant TNF allowed preparation of a globulin fraction by ammonium sulfate precipitation, diisopnopyl fluorophosphate treatment, and dialysis against 10 mM HEPES saline (pH 7.4). Adsorption using washed rabbit red blood cells removed naturally occurring goat hemagglutinating antibodies. When antibody was mixed and incubated with an equal volume of conditioned medium from LPSstimulated rabbit macnophages (TNF source) for 30 mm at 37#{176}C, a 1:400 dilution of antiserum neutralized 2000 U of TNF per ml. The anti-HrTNF contained 40 mg total protein per ml. The L929 tity of TNF (1200 antibody. A 34:1 neutralized TNF course of in vitro
Fig.
2. incubated
Electron with
assay was performed using a known quanU) with varying amounts of the anti-TNF ratio of anti-HrTNF to TNF completely cytolysis of L929 cells (Fig. 1A). The time alveolar m TNF production after LPS
micrographs LEH, showing
of alveolar phagocytosis
macrophages of liposomes
stimulation TNF was to accumulate
et al.
over
the
time L929 24-h
periods assay period
was also documented. after 4 h and continued of LPS
stimulation
(Fig.
1B).
Northern
blot analysis
Alveolar macrophages were incubated with 100 tl of LEH (18 month storage) for 30 mm. Plated cells were washed with RPMI 1640 and stimulated with 1, 10, or 100 ng/ml LPS for 3 h. Activated macrophages were harvested and Northern blot analysis for rabbit tumor necrosis factor mRNA was performed. Total cellular RNA was isolated from harvested alveolar macrophages using the method described by Chomczymski and Sacchi [13]. RNA (10 g per lane) was electrophorcsed in a 1% agnose-formaldehyde gel and transfenred to nylon membranes (Nytran; Scheiched and Schuell). cDNA fragments for rabbit TNF (a gift from Dr. Torao Tshida, Asahi Chemical Industry Co., Tokyo) or f3-actin (a gift from Dr. Sharon Busby, ZymoGenetics, Seattle, WA) were labeled with [3P]dCTP by random priming and used to detect specific transcripts by blot hybridization and autonadiognaphy. trol to ensure
Actin mRNA production equivalent RNA loading
was used as in each lane.
a con-
Analysis of results Values represent the production in untreated
(original magnification x 18,000). into intracellular vacuoles (arrows).
Langdale
for various detectable by
Liposome
(A)
Unstimulated
encapsulated
mean fold increase macnophagcs ±
alveolar
hemoglobin
over basal TNF standard error of
macrophages.
and
(B)
TNF
release
Macrophages
681
the mean (SEM). Wilcoxon signed rank testing for statistical comparison of differences between controls (normal cellular response to LPS stimulation) and macnophages exposed to LEH (various incubation periods and doses of LPS) was penformed. Friedman analysis of variance by ranks was calculated to test for effects of exposure time. The significance level was set at P < .05.
LEH 500
Normal .5 hour 1 hour 3hour
U
-
l 400-I 0)
#1 (stored
J
6 months)
cells
300
C
0)
Cl) Co
RESULTS Treatment 0111:B4 paralleled
of rabbit LPS induced previously
alveolar the production described
macnophages ofTNF dose-response
200
#{149}D 0)
with E. coli in a pattern that curves [14].
100W
(5
0) 5-
C
Electron microscopy demonstrated macrophage phagocytosis of the liposomes when cells were incubated with 100 tl of LEH for 1 h (Fig. 2A and B). LEH phagocytosis alone did not induce TNF release. When m4s pretneated with LEH were stimulated with LPS, significant inhibition of TNF cxpression was observed over a broad dose range of endotoxin, compared to ms pretncated with medium alone and stimulated with equivalent doses of LPS (Fig. 3). This effect was similar for LEH solutions refrigerated for 6 on 18 months. To determine whether the period of exposure to LEH was a variable, similar experiments were performed, incubating alveolar ms with LEH for 30 mm, 1 h, and 3 h. Altering the period of exposure to either stored product did not affect TNF measurements, which remained significantly below control levels for equivalent stimulating doses of endotoxin. TNF suppression was achieved within 30 mm of incubation with LEH. Comparing the three incubation periods for a given dose of LPS, neither solution showed significant differences between cytokine levels (Fig. 4). To determine whether tumor necrosis factor production was a function of the LEH dose, alveolar macrophages were
0
5-
1
10
100
(stored
18 months)
a) >
0
LEH
0)
#2
500
Cl) Co
0) 5-
0 C
400
0
300
‘4-
U-
z I-
200 100
10 LPS (ng/ml) Fig.
4.
production
500.
Effect
of
was
animals.
Altering
did
affect
not
increasing the
the
throughout
the
Friedman
analysis
time
determined time of of
exposure with
of exposure
inhibition
range
of
in duplicate of LPS
variance
(0.5,
to
jzl/ml
macrophage
LEH.
macrophages
1, or 3 h) to a fixed TNF
concentrations by
100
alveolar
dose
production.
(no
significant
TNF from
six
of LEH This
held
difference
by
ranks).
0) C 0) Cl) Co .0
I
400.
Normal LEH
.
cells incubated with 100 or 400 l of LEH for 3 h prior to LPS stimulation. Increasing the LEH dose resulted in further inhibition ofTNF expression with equivalent endotoxin stimulation. This pattern held for both solutions (Fig. 5). Statistically insignificant increases in TNF were observed with increasing LPS concentrations beyond 10 ng/ml (P .08 by Wilcoxon signed rank test), an endotoxin level comparable
#1
a) LEH #2
CS 0) 5-
C
300
5.-
0) > 0
0)
to that found [15]. Inhibition
200
Cl) CS
0) 50 C
all
100
0
I-
0 0
1
10
100
1000
LPS (ng/mI) Fig. 3. Alveolar macrophage of LEH. TNF production was phages from six animals. Cells 18 months) showed a significant normal cells over a dose range trations ( 10 ng/ml). *P
0
C)
Liposome encapsulation of functional hemoglobin molecules shows promise as a universal red cell substitute, offering an efficient means of transporting oxygen and carbon dioxide with an intravascular half-life beyond that characteristic of
C
free
#2 (stored
LEH
a)
18 months)
U)
cci a)
0 0
LL
z I-
100
0
0
100
10
Fig. 5. Effect was
determined
TNF
response
further increasing
of increasing
LEH
in duplicate to
LEH
inhibition the
doses
of macrophage LEH
dose,
with of
3-h
time
alveolar 100
and
TNF
1000
(ng/ml)
LPS
exposure.
macrophages 400
d
are
TNF from
shown.
production/release
production six
animals.
A trend
toward
is associated
with
dose.
The presence of an inhibitory agent in the supernatant from LEH-trcated, LPS-stimulated ms might also account for the lack ofTNF measured by the L929 cytolysis assay. To exclude this possibility, the assay was repeated using conditioned medium from m4s pretreated with 400 tl of LEH (6 month storage) and stimulated with 0, 10, and 100 ng/ml LPS, adding 100 U of TNF to each well of 5 x 10 L929 fibroblasts/well. The effects of exogenous tumor necrosis facton and TNF produced by LEH-treatcd, LPS-stimulated ms on L929 cytolysis were additive (Fig. 6B). These results suggest that TNF expression after LEH phagocytosis was not secondary to an inhibitory agent in the conditioned medium. Because cellular destruction of TNF during the incubation period might also explain the observations, a similar dose of TNF was added to the m4s during pretreatment with LEH and prior to LPS stimulation. TNF measurement increased appropriately, confirming that cellular destruction of the cytokine did not account for the observed decrease in TNF expression (data not shown). As additional controls, alveolar macrophages were treated with liposomes of similar chemical composition in which hemoglobin had not been encapsulated (100 l empty liposomes) in the presence or absence of stroma-free hemoglobin solution (hemoglobin concentration = 6.9 g/dl; methemoglobin = 29%). Despite empty liposomes similar to stimulation
contamination had an inhibitory
that observed (Fig. 6C).
with
with Pseudomonas effect on TNF LEH
after
E.
coli
solutions.
The
impact
of such
a product
on
the liver and lung have been documented [16], the qualitative effects of an agent that alters alveolar macrophage function would be expected to be similar for Kupifer cells and intravasculan pulmonary m4s. In addition, if the neticuloendothelial system were functionally saturated, as might occur with large-dose infusions of LEH, pulmonary macnophagcs might assume a significant role in liposomal clearance. Studies in which pulmonary intravascular m4 phagocytosis of circulating phosphatidylcholinc-cholesterol-phosphatidylsenine liposomes has been confirmed by electron microscopy and indium radiolabeling techniques suggest that pulmonary macrophagcs may indeed play an active role in liposome clearance [6]. Although alveolar macrophages would see only systemic LEH under conditions of transpulmonary capillary leak (for example, adult respiratory distress syndrome genic
after trauma on sepsis), cardiogenic and pulmonary edemas frequently complicate
settings in which a blood substitute useful adjunct to therapy. Therefore, rophages to evaluate effects of LEH phagocytosis is both practical and The appearance of large, clear
endotoxin
et al.
noncandiothe clinical
might be considered a the use of alveolar macon cellular function after relevant. vacuoles in the alveolar
macnophages incubated with LEH (Fig. 2) is consistent with the observations of other investigators who, through the use of various liposome-encapsulated detection probes, have demonstrated that liposomes become concentrated and arc gradually [17, 18]. liposome showed tivity, unaffected
aeruginosa,
expression
Langdale
hemoglobin
reticuloendothelial and immunologic competence, however, has not been fully assessed. In this study, alveolar macnophages were utilized to determine the effects of stored liposome encapsulated bovine hemoglobin solution on macrophage function, as defined by tumor necrosis factor expression with E. coli endotoxin stimulation. Clearance of circulating LEH is presumed to be primarily via hepatic and splenic macnophage phagocytosis [15]. Investigations directed to analyses of these macnophages, however, are limited by isolation techniques that may perturb cell function. By contrast, alveolar macrophages harvested by bronchoalveolar lavage arc minimally affected. Although phenotypic differences between fixed tissue macrophages of
Liposome
degraded In a study
in lysosomal vacuoles designed to address
of phagocytic cells potential toxicity of
phagocytosis, Gonzalez-Rothi and colleagues [17] that alveolar macrophage phagocytic and killing acsurface adherence, and respiratory burst are by incubation with empty liposomes. Functional encapsulated
hemoglobin
and
TNF
release
683
w
A.
LEH
#1 stored
C
20
6 months
LEH #2
stored
1 8 months
I
LEH
washed
LEH
unwashed
Normal
20
“5
4)
0 C
(5 4)
0
60
cells
Empty
liposomes
Empty
liposomes
+
hemoglobin
Free
Cl)
C
0 .0
4)
10
50
10
0
a) Co
a)
4) C
40
C
0
0
U-
z
0
10
100
5a)
1000
0
10
100
30
1000
0
4-
LPS Dose
LPS Dose
(ng/ml)
(ng/mI)
a) Cl) Co
B.
o
LEH+TNF
C.) C
D 0
40
!
20
5. a)
ILEH
10
U-
z
30
C
U
I-.
0
U-
z
1
20
4-
LPS
Dose
(ng/ml)
10
100
10
LPS Dose
Fig.
6. (A)
remove tion
To
the
LEH
in the
altered
(B)
and
to
chemical that
the
h of LPS
of LEH
for
of
LEH,
To
the
test
for
of
ofTNF
liposomes
previously
(ng/mI)
by
LEH,
equivalent
period that
presence
incubated
This
occurred
with
containing
been
a
or
of
the
stimulation
for
cells
3 h.
were
of the
responsible
conditioned
for
medium
the
Cells
not
were
washed
macrophages
observed
produced
when
washed and
three
LPS
was
to produce
lack
of TNF
alveolar
times added
TNF
was
expression
macrophages
with
RPMI
to allow not
from were
to
stimula-
appreciably LEH-treated
incubated
with
consistent
stimulation
not
over
The
absence
of
hemoglobin
as
range
to the
inhibitory
free
such
cytokines
the
secondary
of
LPS
presence
effect
on
ofan the
inhibitory
expression
for
both
LEH
agent. of TNF
(C) by
preparations. Empty
of LEH with alveolar E. coli endotoxin-induced This effect was indepen-
These
liposomes
LPS-stimulated
results
of comparacells
was
similar
solution.
prostaglandin E2, and leukotnienes, which play in the immune (cellular and humoral) and responses to injury and infection [27]. Inhibition pression after large-volume LEH infusion and
have
reported.
present study, incubation was shown to inhibit of tumor necrosis factor.
LPS
not
LEH
of L929 cytolysis assay. Because limited quantities of The difference between TNF measured in the medium
was
protein,
complex
expression
treated
added to the medium at the time only with the LEH I preparation.
macrophages.
presence
was
with
similarly assay.
LPS in
was
to LEH
alveolar the
the
agent
of TNF
incubated
containing to L929
of
TNF was performed
exposure
in
prior
inhibiting
quantity after
were
of time
of LPS. were
were
wells
adsorption
of an
of a known
macrophages
Additional
suggesting
macrophage
alveolar
stimulation.
expression
LEH.
on
In the macrophages expression
an
the
addition
lack by
hemoglobin,
not
to 20
composition
induced
effects
adsorption
stimulated with varying doses were available, these experiments
without that
LPS
presence
and then 2 material
indicate ble
prior
the
macrophages.
with
for
presence
h
LEH LEH
test
critical roles nonimmunc ofTNF cxclearance by
of fungal infections have found no effects [2, 19-21], infusion of larger are known to suppress reticuloendothelial function [22]. Since the diffuse organ injury associated with severe trauma and sepsis is thought to be the result of an uncontrolled inflammatory response [22], our demonstration of inhibited TNF expression by macrophages incubated with a potential red cell substitute such as LEH has important clinical implications. Tumor necrosis factor has been implicated as a central inflammatory mediator in the pathogenesis of gram-negative
fixed tissue macrophages may result in modulation of the overall severity of the inflammatory response to trauma and/or sepsis. In addition to inducing the production of TNF and other cytokines, endotoxin is known to initiate a cascade of metabolic events, including the mobilization of peripheral energy stores in the form of lipoproteins. Cytokines produced by activated m4s further increase available triglyceride fatty acids by suppressing synthesis of lipoprotein lipase [28]. It has been postulated that the hyperlipopnoteincmia of sepsis may represent a host defense mechanism, aimed at detoxification of circulating endotoxin [29]. Binding to cholesterol-rich lipoproteins has been shown to decrease LPS toxicity to endothelial cells in vitro and prevent death in endotoxinsensitive mice [30-32]. Harris and associates reported that incubation of very low density lipoprotcins (VLDLs) and chylomicrons with endotoxin prior to infusion into mice pro-
shock, acting in part to alter the hemostatic and inflammatory properties ofvasculan endothclial cells [23] and circulating inflammatory cells [24-26]. TNF also induces the biosynthesis or release of other inflammatory mediators, including interleukin-1, procoagulants, thromboxanc B2,
tected against endotoxin-induced death, presumably by adsorptive detoxification of LPS [33]. These data support a potentially protective role for lipids in gram-negative sepsis. Chemically, cholesterol-based liposomes have much in common with naturally circulating lipoproteins. One might
dent of the period of LEH, however, Although previous volume
infusions
of exposure further studies of
liposomes
to LEH. inhibited evaluating as
drug
Increasing the dose TNF expression. the use of smallcarriers
for
cancer
pa-
tients and the treatment shortor long-term volumes of liposomes
684
Journal
of
Leukocyte
Biology
Volume
52,
December
1992
LPS
LPS
Inhibition ofTNF expression may lation as well as gene transcription, demonstrated with dexamethasone
LEH
+
11 0
0
00 ‘-0
0
that LEH may manner. Altered
00
an equally attractive observed inhibition tosis. Liposome-encapsulated several modifications
las
TNF
las
Fig.
7.
TNF
overall resolved,
28S Induction
of TNF
mRNA
gene
in macrophages
transcription. incubated
untreated cells. Blots were probed dized to a fl-actin probe to assess tration zg
of
of
TNF
LPS
LPS
(0-100
with
and
accumulates
ng/ml). without
with
lOOX
ism
of
cells
for both
TNF
protein
LPS
doses.
LEH
in a similar would provide
alternative mechanism of TNF expression after
to explain the LEH phagocy-
hemoglobin to achieving
to undergo formulation.
prior
is likely its final
Comparing
the
protein endotoxin
lanes
loading
This
suggests
of
30
interest,
is similar.
stimulation
ofTNF than
mm
and at each
in both
and
versus
rehybriconcen-
10 and
100
Message treated
and
recoun-
mechan-
inhibition.
therefore predict that liposomes would exhibit a protective effect with endotoxic challenge similar to that seen with VLDLs on chylomicrons. Our data suggest an alternative mechanism by which endotoxin-induced TNF release may be inhibited. Alveolar ms incubated in the presence of LEH produced significantly less TNF after stimulation with endotoxin than cells not exposed to LEH. Because comparable levels of TNF were produced from LPS-stimulated cells, whether or not the liposomes were removed prior to the addition of the endotoxin, adsorption of LPS does not appear to be responsible for the observed inhibition of TNF exprcssion. By contrast, Northern blot analysis of total cellular RNA obtained from m4s treated with medium or medium containing LEH prior to stimulation with cndotoxin showed an accumulation of TNF-spccific transcripts under both conditions. The appearance of TNF-spccific mRNA after pretreatment with LEH suggests that LEH inhibition of LPS-activatcd alveolar macnophages is an intracellular, posttranscriptional event. It is not known whether the posttranscniptional modulation of TNF expression by LEH is due to impaired translation or altered intracellular transport and release of a functional protein product. Eukaryotic mRNAs are modified after transcription, in part by the addition ofa 3’ poly(A) tail that may affect the regulation of mRNA translation [34]. It is tempting to speculate that LEH interferes with this modification of TNF transcripts or with the action of the 3’ poly(A) tail. The promoter and 3-untranslated regions of the TNF gene contain sequences that independently regulate the response of mononuclear cells to endotoxin. Beutler and colleagues have postulated that the addition of endotoxin mobilizes pooled intracellular TNF mRNA for translation in addition to stimulating biosynthesis of additional message.
Langdale
of the of
use
red cell substitute. liposome-encapsulated
Once
these issues hemoglobin
are in
endotoxemia.
ACKNOWLEDGMENTS
for
mRNA were from stimulated
a posttranscriptional
safety
hemorrhagic shock or trauma resuscitation may have the additional advantage of blunting a potentially deleterious inflammatory response to subsequent gram-negative sepsis
of LPS-induced for
for TNF-specific transcripts RNA loading among wells
LEH,
increasing
Comparison with
untreated cells. However, greater concentrations vered from stimulated cells incubated with LEH treated
TNF mRNA translation transport and release
Biochemical questions pertinent to product safety are under investigation and include accurate measurement of LEH stability over time, the importance of increasing methemoglobin levels, and choice ofstorage buffer. Our data affirm that in vivo evaluation of an animal’s ability to respond to an cndotoxin challenge after exposure to largevolume infusions will be essential to an evaluation of the
28S
3-Actin
inhibit protein
occur at the level of transan effect that has been [35, 36]. It is possible
et a!.
The
authors
wish
to thank
Dianne
Neil
and
Greg
Hahncl
their technical expertise and assistance. Supported Health Sciences Research and Development Department of Veterans Affairs.
for
by the Service,
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