Liposome-encapsulated factor

release

drome such

of Surgery,

Macrophages response

through the as tumor

that

production necrosis

alveolar

macrophages

V. Maier,

University

contribute characterizes

of

to

of inflammatory factor (TNF).

Washington

Lynne and

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

cytokines Liposomecell subIn these with of TNF stimulabut in-

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

the systemic the sepsis syn-

encapsulated hemoglobin (LEH), a potential red stitute, is cleared by fixed tissue macrophages. studies, in vitro incubation of alveolar macrophages stored LEH was shown to inhibit the expression induced by endotoxin (lipopolysaccharide, LPS) tion. This effect was dependent on LEH dose

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

Cell preparation

1000

1200 I 000 Cl)

.-

C U-

z

400

I200.

ng/ml.

and experimental

design 0.0

The with

ease with which minimal perturbation

population

ideally

alveolar macnophagcs ofcellular function

suited

to in vitro

analysis

0.5

are harvested made this cell of the

effects

tO

Ratio

1.5

2.0

34

of Anti TNF to TNF

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

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

and

was

LPS-stimulated

tion

final

at 4 h and was

1%

observed

BSA

cytolysis by

antibody

ize the cytokine mences

L929

confirmed

TNF-specific by

the

was

at a ratio continues

dilution

the

(A) the

in varying

of 1:34.

alveolar over

added

(B)

20

L929

Specificity

assay

of the

using

dilutions

1200 and

measurement

found

to accumulate.

No

diminution

at

-80#{176}C for

and

absence

assay

of TNF.

A

to neutral-

ofTNF

in

h of

the

L929

U

macrophages 24

24

(hrs)

assay.

performing

16

produced

of

serum

ofTNF

cornproduc-

stimulation.

stored

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 Rockville, MD) were pretneated with actinomycin Chemical Co., St. Louis, MO) at 2 tg/ml for RPMI 1640 supplemented with 5% horse serum Laboratories, Logan, UT) and added to the serial of the conditioned medium at 5 x 10’” cells/well.

with

as

1992

assay of fibroblasts Collection, D (Sigma 15 mm in (Hyclonc dilutions After 24 h

of incubation at 37#{176}Cwith 5% C02, the cells were fixed and stained with 0.1% crystal violet in 20% methanol. Dried monolayers were solubilized in 0.1 M sodium citrate in 50% ethanol and the absorbance was read at 550 nm. One unit of TNF 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 Chomczymski and Sacchi [13]. RNA (10 g electrophorcsed in a 1% agnose-formaldehyde fenred to nylon membranes (Nytran; Scheiched

described by per lane) was gel and transand 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


0

LEH

0)

#2

500

Cl) Co

0) 5-

0 C

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 TNF with given ences To was a

200

400

0

300

‘4-

U-

z I-

200 100

levels for equivalent stimulating doses of endotoxin. suppression was achieved within 30 mm of incubation LEH. Comparing the three incubation periods for a dose of LPS, neither solution showed significant differbetween cytokine levels (Fig. 4). determine whether tumor necrosis factor production function of the LEH dose, alveolar macrophages were

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

#2 (stored

LEH

a)

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

18 months)

U)

cci a) C) C

free

0 0

LL

z I-

100

0

0

100

10

Fig. 5. Effect was

determined

TNF

response

further

of increasing to

inhibition

increasing

LEH

in duplicate

the

LEH

doses

of macrophage LEH

dose,

with of

3-h

time

alveolar 100 TNF

and

1000

(ng/ml)

LPS

exposure.

macrophages 400

d

are

production/release

TNF from

shown.

production six

A trend

animals. 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 contamination with Pseudomonas aeruginosa, empty liposomes had an inhibitory effect on TNF expression similar to that observed with LEH after E. coli endotoxin stimulation

(Fig.

6C). Langdale

hemoglobin

solutions.

The

impact

of such

a product

on

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

et al.

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 after trauma on sepsis), cardiogenic and noncandiogenic pulmonary edemas frequently complicate the clinical 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

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 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

C

20

#1 stored

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

the

(B)

and then 2 material and

to

that

chemical that

the

h of LPS

of LEH

for

of

LEH,

To

the lack

of

by

test

for

LEH,

the

period that

incubated

This

occurred

with

containing

been

a

of

or

of

the

stimulation

for

cells for

medium

3 h.

were

of the

responsible

conditioned

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

LEH with alveolar endotoxin-induced effect was indepenIncreasing the dose TNF expression. the use of small-

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.

In the present study, incubation of macrophages was shown to inhibit E. coli expression of tumor necrosis factor. This dent of the period of exposure to LEH. of LEH, however, further inhibited Although previous studies evaluating

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

to L929

agent

of TNF

incubated

containing

the

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

equivalent presence

macrophage

alveolar

stimulation.

expression

LEH.

on

previously

an

(ng/mI)

by

suggesting

addition ofTNF

liposomes

hemoglobin,

not

to 20

composition

induced

effects

adsorption

stimulated with varying doses were available, these experiments

without

indicate ble

prior

LPS

presence

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

volume

infusions

of

liposomes

as

drug

carriers

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

LPS

TNF

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

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

and

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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