A choline-rich diet improves survival in a rat model of
endotoxin shock
Chantal A.
Rivera,
Michael D.
Wheeler,
Nobuyuki
Enomoto, and
Ronald G.
Thurman
Laboratory of Hepatobiology and Toxicology, Department of
Pharmacology, University of North Carolina, Chapel Hill, North
Carolina 27599
 |
ABSTRACT |
This study
investigated whether dietary choline can prevent endotoxin shock.
Female Sprague-Dawley rats fed chow or chow plus choline chloride
(0.025-0.4%) for 3 days were given lipopolysaccharide (LPS) via
the tail vein. Eighty-three percent and 56% of chow-fed rats survived
after 2.5 or 5.0 mg/kg LPS, respectively. Choline increased survival in
a dose-dependent manner, with maximal effects observed at 0.4%; this
dose of choline prevented mortality completely after 2.5 or 5 mg/kg
LPS. Choline also improved the microscopic appearance of the lungs and
blunted increases in serum aspartate aminotransferase levels.
Intracellular Ca2+ was monitored
in liver and lung macrophages during LPS exposure. Ca2+ increases in macrophages from
choline-fed rats were blunted by 40-60% compared with chow-fed
controls. Feeding choline also blunted tumor necrosis factor-
production. Feeding glycine, which prevents macrophage activation via a
chloride channel, in addition to choline was even more effective than
feeding choline alone, suggesting that glycine and choline act via
distinct sites. These data are consistent with the hypothesis that
choline diminishes endotoxin shock by preventing macrophage activation.
Kupffer cells; lipopolysaccharide; tumor necrosis factor-
 |
INTRODUCTION |
ENDOTOXIN is a cell wall component of gram-negative
bacteria cleared from the systemic circulation largely by Kupffer
cells, the resident macrophages of the liver. It has been shown that both circulating and fixed mononuclear phagocytes are activated by
endotoxin and release toxic cytokines that mediate injury and mortality
observed during exposure to endotoxin. Administration of a lethal dose
of endotoxin to rats (endotoxin shock) results in severe hypotension
and multiple organ system failure. Liver and lung injury normally
appear within 8 h and rats usually die of respiratory failure in
12-24 h. Because mortality is believed to occur due to respiratory
failure, it is likely that release of toxic mediators from activated
lung macrophages plays a pivotal role in the manifestation of endotoxin
shock.
Using a model of intravenous endotoxin administration, Nolan and Ali
(13) demonstrated that mortality due to endotoxin shock was increased
significantly in rats fed a choline-deficient diet. On the basis of
this finding, it was hypothesized that a choline-rich diet may prevent
endotoxin shock. The results presented here support this hypothesis and
demonstrate that choline blunts macrophage activation due to endotoxin
[lipopolysaccharide (LPS)].
 |
METHODS |
Dietary treatment.
Female Sprague-Dawley rats (250-275 g) were fed standard
laboratory chow (Prolab RMH 3000; Agway, Syracuse, NY), chow plus choline chloride (0.025-0.4%), chow plus glycine (5%), or chow plus 5% glycine and 0.4% choline chloride for 3 days. Rats were given
free access to water and were maintained on a 12:12-h light-dark cycle.
All rats were given adequate care in accordance with institutional guidelines.
Endotoxin treatment.
After rats were fed for 3 days on the diets described above, LPS
(Escherichia coli serotype O111:B4;
Sigma Chemical, St. Louis, MO) suspended in pyrogen-free saline was
injected via the tail vein. The dose of LPS ranged from 2.5 to 20 mg/kg. Survival was assessed after 24 h, and in some experiments, blood
and tissue samples were collected 8 h after LPS injection. Serum was
stored at
20°C for later measurement of aspartate
aminotransferase (AST) activity by standard enzymatic methods (1). Lung
samples were fixed in phosphate-buffered Formalin and embedded in
paraffin. Blind evaluation of hematoxylin and eosin-stained sections
was performed.
Kupffer cell isolation.
Kupffer cells were isolated from rats fed chow or choline-supplemented
diets by collagenase digestion and differential centrifugation as
described previously (16). Briefly, the portal vein was cannulated, and
livers were perfused with Ca2+-
and Mg2+-free Hanks' balanced
salt solution (HBSS; 37°C) for 5 min. Perfusion was continued with
HBSS containing 0.025% collagenase type IV (Sigma) for ~5 min. When
digestion appeared complete, the liver was removed, placed in a beaker
containing collagenase buffer, and cut into small pieces. The
suspension was filtered through nylon gauze and centrifuged for 10 min
at 450 g at a temperature of 4°C.
The cell pellet was resuspended in HBSS, and parenchymal cells were
removed by centrifugation at 50 g for
3 min. The nonparenchymal cell fraction was washed twice with buffer.
Kupffer cells were isolated by centrifugation through Percoll
(Pharmacia, Uppsala, Sweden) at 1,000 g for 15 min. Viability was determined
by trypan blue exclusion and was >90%. Cells were seeded on glass
coverslips, and culture medium was exchanged after 1 h to remove
nonadherent cells. Dulbecco's modified Eagle's medium (DMEM)
supplemented with 10% fetal bovine serum, 100 U/ml penicillin G, and
100 mg/ml streptomycin sulfate was used. Purity was determined from the percentage of cells that engulfed latex beads and was found to be near
100%. Cells were cultured for ~24 h before experiments.
Alveolar macrophage isolation.
Female Sprague-Dawley rats were fed chow or chow + 0.4% choline for 3 days. Alveolar macrophages were isolated by lavage with sterile PBS via
an intratracheal cannula as described elsewhere (2). Cell suspensions
were centrifuged at 500 g for 7 min, and red blood cells were lysed with 0.15 M
NH4Cl. Cells were resuspended in
buffer containing (in mM) 145 NaCl, 5 KCl, 10 HEPES, 5.5 glucose, and 1 mM CaCl2 (pH 7.4). Viability
assessed by trypan blue exclusion was >90%.
Measurement of
[Ca2+]i.
The fluorescent Ca2+ indicator dye
fura 2 was used to measure intracellular
Ca2+ concentration
([Ca2+]i)
as detailed previously (10). Briefly, Kupffer cells or alveolar macrophages plated on coverslips were incubated in modified Hanks' buffer containing 0.03% Pluronic F-127 (BASF; Wyandotte,
Wyandotte, MI) at room temperature for 1 h. Changes in fluorescence
intensity of fura 2 at excitation (340 and 380 nm) and emission (520 nm) wavelengths were monitored in individual cells, and values were corrected for system noise and autofluorescence as described in detail
elsewhere (10).
Tumor necrosis factor-
measurement.
For determination of tumor necrosis factor-
(TNF-
) in serum, LPS
(10 mg/kg in pyrogen-free saline) was injected via the tail vein. A
cannula was implanted in the jugular vein, and blood samples were
collected after 0, 15, 30, 45, 60, 90, 120, and 180 min. To each
100-µl blood sample, 30 µl of aprotinin (Sigma) were added. For in
vitro TNF-
measurement, alveolar macrophages were cultured on
24-well plates at a density of 5 × 105 cells/well, and medium was
exchanged after 1 h to remove nonadherent cells. The culture medium
used was DMEM. In some experiments choline was added to the culture
medium at a final concentration of 1 mM. After 24 h, the culture medium
was replaced with fresh DMEM containing 1 µg/ml LPS, and cells were
incubated in the presence or absence of 1 mM choline for an additional
4 h. TNF-
was measured in serum and culture medium samples by ELISA
(Genzyme, Cambridge, MA).
Statistical analysis.
Results are means ± SE. Significance was determined using
Student's t-test, Mann-Whitney's
rank-sum test, or Kruskal-Wallis ANOVA on ranks where appropriate. The
Fisher's exact test was used to determine significance in the
mortality studies; P < 0.05 was
selected before the study as the level of significance.
 |
RESULTS |
Effect of dietary choline on survival after LPS injection.
There were no significant differences in the average daily consumption
of chow or choline-supplemented diets (18.1 ± 1.3 and 18.5 ± 1.3 g, respectively). Body weights for rats in the two groups were also
similar (chow, 264 ± 4 g; choline, 265 ± 3 g). After 3 days on
the diets, rats received LPS (2.5-20 mg/kg) injected via the tail
vein. Of rats fed chow, 17% died within 24 h after 2.5 mg/kg LPS, 56%
died at the 5 mg/kg dose, 83% at 10 mg/kg, and 100% at 20 mg/kg (Fig.
1). Death usually occurred 8-12 h
after LPS, and surviving animals showed improvement after 24 h. The addition of 0.4% choline to the diet increased survival to 100% after
injection of 2.5 or 5.0 mg/kg LPS (P < 0.05), whereas 44% of rats survived at the 10 mg/kg dose (Fig. 1).
To determine the maximal effective dose of choline, the amount of
choline added to the diet was varied between 0.025% and 0.4%. Choline
improved survival after 5 mg/kg LPS in a dose-dependent manner and was maximally effective at 0.4% (Fig. 2).

View larger version (13K):
[in this window]
[in a new window]
|
Fig. 1.
Effect of choline on lipopolysaccharide (LPS)-induced mortality. Rats
were fed chow or chow + 0.4% choline for 3 days. LPS was injected into
the tail vein at doses indicated, and mortality was assessed after 24 h. Fractions presented are survivors/total.
* P < 0.05 (Fisher's exact
test).
|
|

View larger version (12K):
[in this window]
[in a new window]
|
Fig. 2.
Effect of varying dietary choline on survival after LPS. Rats were fed
chow with various amounts of added choline chloride for 3 days. LPS (5 mg/kg) was injected via the tail vein, and survival was assessed after
24 h. Fractions represent survivors/total.
|
|
Effects of dietary glycine and choline are additive.
In previous studies, dietary supplementation with glycine blunted
Kupffer cell and alveolar macrophage activation and increased survival
due to endotoxin shock (9, 24). Glycine inhibits Kupffer cell
activation due to LPS by hyperpolarization of the plasma membrane by
activating a glycine-gated
Cl
channel (10). To
determine if the effects of glycine and choline were additive, rats fed
chow, chow supplemented with glycine or choline, or chow supplemented
with a combination of glycine and choline were given a lethal dose of
LPS (10 mg/kg) as described above. Under these conditions, 17% of
chow-fed and 43% of choline-fed rats survived (Table
1). Glycine alone also improved survival by
50%; however, given together, glycine and choline increased survival
to 100%. Since the effects of glycine and choline are additive in this
model, it is likely that choline acts at a site distinct from the
Cl
channel activated by
glycine.
Effect of dietary choline on serum AST and histology after LPS.
Blood samples were collected 8 h after injection of 5 mg/kg LPS. Basal
serum AST values were 65 ± 33 and 55 ± 18 U/l in the chow and
chow + 0.4% choline groups, respectively (Fig.
3). Injection of LPS increased AST to 846 ± 146 U/l in chow-fed animals; however, this increase was blunted
significantly by feeding choline, with values only reaching 163 ± 22 U/l (P < 0.05). Representative
photomicrographs of typical lung samples collected 8 h after treatment
of rats with 5 mg/kg LPS are shown in Fig.
4. Increased cellularity, alveolar filling,
and inflammation were observed after LPS exposure in rats fed chow
(Fig. 4A). Lung pathology was
improved markedly by choline (Fig.
4B).

View larger version (10K):
[in this window]
[in a new window]
|
Fig. 3.
Effect of choline on LPS-stimulated serum aspartate aminotransferase
(AST) levels. Conditions are as described in Fig. 1 legend. Blood
samples were collected 8 h after injection of 5 mg/kg LPS.
* P < 0.05 compared with
control. # P < 0.05 compared
with chow + LPS (Kruskal-Wallis ANOVA on ranks;
n = 4).
|
|

View larger version (90K):
[in this window]
[in a new window]
|
Fig. 4.
Effect of choline on lung histology after LPS injection. Conditions are
as described in Fig. 1 legend. Representative lung specimens were
collected for histology 8 h after LPS injection (5 mg/kg) and
stained with hematoxylin and eosin. A:
chow-fed rat. B: chow + 0.4%
choline-fed rat. Original magnification, ×40.
|
|
Effect of dietary choline on changes in
[Ca2+]i
in cultured macrophages.
Increased
[Ca2+]i
is an essential precursor event for the production of proinflammatory
cytokines (17) and was used here as a marker of Kupffer cell and
alveolar macrophage activation (Figs. 5A and
6A).
Intracellular Ca2+ was monitored
fluorometrically in individual cells isolated from chow- or choline-fed
rats. After the addition of 10 µg/ml LPS to the culture medium of
Kupffer cells isolated from chow-fed rats,
[Ca2+]i
increased rapidly, reaching a peak value of 241 ± 16 nM
within 100 s, followed by a decline to basal levels within 200 s (Fig. 5A).
However, when Kupffer cells were isolated from rats fed 0.4% choline
for 3 days, the increase in
[Ca2+]i
due to LPS was only ~60% as large as the response in Kupffer cells
isolated from chow-fed rats (Fig.
5B). Similarly, LPS-stimulated [Ca2+]i
reached peak values of 207 ± 14 nM in alveolar macrophages from
chow-fed rats (Fig. 6A).
[Ca2+]i
in alveolar macrophages isolated from rats fed a choline-rich diet only
reached 72 ± 7 nM and was significantly lower than the response
observed in cells from rats fed chow (Fig. 6,
A and
B). The addition of 10 mM choline
chloride to the culture medium 6 or 24 h before LPS did not alter
increases in Ca2+ caused by LPS
(data not shown).

View larger version (10K):
[in this window]
[in a new window]
|
Fig. 5.
Effect of choline on LPS-stimulated intracellular
Ca2+ concentration
([Ca2+]i)
in isolated Kupffer cells.
[Ca2+]i
levels in cultured Kupffer cells from chow- or chow + choline-fed rats
were measured fluorometrically using fura 2 as described in
METHODS. Addition of LPS (10 µg/ml)
is denoted with arrows. A:
representative traces. B: mean ± SE of peak
[Ca2+]i
following LPS. *** P < 0.001, Student's t-test;
n = 4 rats/group.
|
|

View larger version (12K):
[in this window]
[in a new window]
|
Fig. 6.
Effect of choline on alveolar macrophages. Alveolar macrophages were
isolated from chow- or choline-fed rats.
A: representative traces of
[Ca2+]i
measured fluorometrically as described in
METHODS.
B: average peak increase in
[Ca2+]i.
* P < 0.05, Mann-Whitney's
rank-sum test. C: alveolar macrophages
were cultured in presence of 1 µg/ml LPS as detailed in
METHODS. Tumor necrosis factor-
(TNF- ) was measured by ELISA.
* P < 0.05 using
Kruskal-Wallis ANOVA on ranks.
|
|

View larger version (37K):
[in this window]
[in a new window]
|
Fig. 7.
Schematic representation of the hypothesized role of choline in the
prevention of lung damage during endotoxin shock. LPS activates
macrophages to release toxic mediators such as TNF- , which results
in lung injury. Mortality after LPS usually results within 8-12 h
and is most likely caused by respiratory failure. Because choline
blunts the increase in
[Ca2+]i
and the release of TNF- from alveolar macrophages stimulated with
LPS, it is hypothesized that choline alters the signaling cascade
triggered by LPS binding to receptors on macrophages, possibly by
increasing membrane phosphatidylcholine (PC) content. By preventing
macrophage activation and the release of TNF- , choline significantly
diminishes lung injury and improves survival. LBP, LPS binding protein;
VDCC, voltage-dependent Ca2+
channel; PI, phosphatidyinositol; DAG, 1,2-diacylglycerol;
IP3, inositol 1,4,5-trisphosphate.
|
|
Effect of dietary choline on TNF-
production.
Serum TNF-
was measured at various times after injection of 10 mg/kg
LPS. In chow-fed rats, serum TNF-
began to increase 45 min after LPS
and reached peak values of 5,369 ± 1,378 pg/ml at 90 min. Choline
had no effect on the increase in serum TNF-
due to LPS, with peak
values reaching 4,475 ± 967 pg/ml at 90 min. To determine the
effect of choline on alveolar macrophages, cells were isolated from
chow- or choline-treated rats and exposed to LPS in the culture medium
(Fig. 6C). Alveolar macrophages
isolated from chow-fed rats produced TNF-
at a rate of 1,018 ± 190 pg · 5 × 105
cells
1 · 4 h
1. Feeding rats a
choline-rich diet before cell isolation significantly diminished
TNF-
production (543 ± 158 pg · 5 × 105
cells
1 · 4 h
1); however,
LPS-stimulated production of TNF-
was not diminished when choline
was added in vitro to the culture medium of cells isolated from
chow-fed rats (1,200 ± 130 pg · 5 × 105
cells
1 · 4 h
1).
 |
DISCUSSION |
Dietary choline reduces tissue injury and mortality in the rat.
Because choline deficiency enhances injury and mortality due to
endotoxin shock (13), it was hypothesized that dietary supplementation with choline would diminish these effects. Standard chow diet contains
~0.12% choline. In the present study, rats were fed diets supplemented with 0.4% choline for 3 days before endotoxin was given.
The addition of choline prevented mortality completely at a dose of LPS
sufficient to cause death in 50% of rats fed standard chow diet (Fig.
1). The protective effect of choline against mortality was associated
with a marked reduction in lung injury (Fig. 5). Because mortality due
to endotoxin shock is believed to result from respiratory failure in
this model, these data are consistent with the hypothesis that choline
improves survival by preventing lung pathology.
How does choline prevent mortality?
Previously, it has been shown (9) that dietary supplementation with
glycine blocks endotoxin-induced injury and subsequent mortality. The
protective effect of glycine was associated with smaller LPS-induced
increases in
[Ca2+]i
in isolated Kupffer cells and alveolar macrophages as well as with a
decrease in TNF-
production (1, 24), a
Ca2+-dependent event (22). Glycine
blunted activation when added to the diet or when added directly to the
culture medium of isolated cells by stimulating the influx of
Cl
, thus hyperpolarizing
the cell membrane (10). As described in this study, feeding a diet rich
in both glycine and choline was even more effective in preventing
mortality than feeding choline or glycine alone (Table 1). In contrast
to glycine, in vitro treatment of macrophages with choline did not
prevent LPS stimulation of
[Ca2+]i
or TNF-
. These findings suggest that the protective mechanism against endotoxin shock observed after dietary supplementation with
choline is distinct from glycine.
Dietary choline inhibits macrophage activation.
A previous study (8) demonstrated that macrophage activation by LPS was
associated with an increased rate of phosphatidylcholine hydrolysis via
a phospholipase-dependent mechanism. Hydrolysis of phosphatidylcholine
by phospholipase C and D generates 1,2-diacylglycerol (DAG) (3). Recent
experiments (26) have shown that DAG formation and protein kinase C-
activation correlate with the activation of nuclear factor-
B, a
transcription factor necessary for production of many cytokines,
including TNF-
. Cytokines contribute to injury and mortality
observed during LPS exposure (20). For example, circulating levels of
TNF-
and interleukin-1 (IL-1) increase rapidly after LPS exposure
(4). Treatment of rats with TNF-
mimics the changes in lipid
metabolism and mortality caused by LPS (12, 18), whereas antibodies
directed against TNF-
prevent these effects (6, 19). Although
administration of IL-1 can reproduce many of the changes in lipid
metabolism associated with LPS exposure, antibodies directed against
IL-1 were not effective (7, 12). Therefore, it is likely that
production of TNF-
is of primary importance in the mechanism of
endotoxin shock.
One possible explanation for the findings presented here is that excess
choline supplied in the diet could increase the ratio of
phosphatidylcholine to polyphosphoinositides in the cell membrane (8).
This could prevent signaling events downstream of LPS binding to
receptors on macrophages, since phosphoinositides, but not
phosphatidylcholine, lead to increases in
Ca2+ necessary for macrophage
activation (3). Alternatively, LPS decreases membrane fluidity, an
effect reversed by the addition of phosphatidylcholine (11). Supplying
excess choline in the diet could alter intracellular signaling due to
LPS by enhancing phosphatidylcholine resynthesis and maintaining proper
membrane fluidity (see Fig. 7). Data presented here demonstrate that
the LPS-stimulated increase in intracellular
Ca2+ was blunted by about 50% in
Kupffer cells and alveolar macrophages isolated from choline-fed rats
(Figs. 5 and 6). Furthermore, TNF-
production by alveolar
macrophages was diminished ~50% by choline (Fig.
6C). Although the exact mechanism
underlying the protective effects of choline is not yet clear, the
findings reported here strongly support the hypothesis that choline
interferes with the intracellular signaling cascade triggered by LPS.
 |
ACKNOWLEDGEMENTS |
This study was supported, in part, by National Institutes of Health
Grants AA-03624 and ES-04325 and by a gift from Novartis Nutrition
Research AG.
 |
FOOTNOTES |
Portions of this work have been published previously in abstract form
(21).
Address for reprint requests: R. G. Thurman, CB# 7365, Faculty
Laboratory Office Building, Univ. of North Carolina, Chapel Hill, NC
27599-7365.
Received 14 August 1997; accepted in final form 22 May 1998.
 |
REFERENCES |
1.
Bergmeyer, H. U.
Methods of Enzymatic Analysis. New York: Academic, 1988.
2.
Bhat, M., Y. Rojanasakul, S. L. Weber, J. Y. C. Ma, V. Castranova, D. E. Banks, and J. K. H. Ma.
Fluoromicroscopic studies of bleomycin-induced intracellular
oxidation in alveolar macrophages and its inhibition by taurine.
Environ. Health Perspect. 102, Suppl. 10: 91-96, 1994.
3.
Canty, D. J.,
and
S. H. Zeisel.
Lecithin and choline in human health and disease.
Nutr. Rev.
52:
327-339,
1994[Medline].
4.
Chensue, S. W.,
P. D. Terebuh,
D. G. Remick,
W. E. Scales,
and
S. L. Kunkel.
In vivo biologic and immunohistochemical analysis of interleukin-1
,
and tumor necrosis factor during experimental endotoxemia: kinetics, Kupffer cell expression, and glucocorticoid effects.
Am. J. Pathol.
138:
395-402,
1991[Abstract].
5.
Decker, K.
Biologically active products of stimulated liver macrophages (Kupffer cells).
Eur. J. Biochem.
192:
245-261,
1990[Medline].
6.
Eastin, C. E.,
C. J. McClain,
K. Lee,
G. J. Bagby,
and
R. K. Chawla.
Choline deficiency augments and antibody to tumor necrosis factor-
attenuates endotoxin-induced hepatic injury.
Alcohol. Clin. Exp. Res.
21:
1037-1041,
1997[Medline].
7.
Fischer, E.,
M. A. Marano,
A. E. Barber,
A. Hudson,
K. Lee,
C. S. Rock,
A. S. Hawes,
R. C. Thompson,
T. J. Hayes,
T. D. Anderson,
W. R. Benjamin,
S. F. Lowry,
and
L. L. Moldawer.
Comparison between effects of interleukin-1
administration and sublethal endotoxemia in primates.
Am. J. Physiol.
261 (Regulatory Integrative Comp. Physiol. 30):
R442-R452,
1991[Abstract/Free Full Text].
8.
Grove, R. I.,
N. J. Allegretto,
P. A. Kiener,
and
G. A. Warr.
Lipopolysaccharide (LPS) alters phosphatidylcholine metabolism in elicited peritoneal macrophages.
J. Leukoc. Biol.
48:
38-42,
1990[Abstract].
9.
Ikejima, K.,
Y. Iimuro,
D. T. Forman,
and
R. G. Thurman.
A diet containing glycine improves survival in endotoxin shock in the rat.
Am. J. Physiol.
271 (Gastrointest. Liver Physiol. 34):
G97-G103,
1996[Abstract/Free Full Text].
10.
Ikejima, K.,
W. Qu,
R. F. Stachlewitz,
and
R. G. Thurman.
Kupffer cells contain a glycine-gated chloride channel.
Am. J. Physiol.
272 (Gastrointest. Liver Physiol. 35):
G1581-G1586,
1997[Abstract/Free Full Text].
11.
Lui, M. S.,
S. Ghosh,
and
Y. Yang.
Change in membrane lipid fluidity induced by phospholipase A activation: a mechanism of endotoxic shock.
Life Sci.
33:
1995-2002,
1983[Medline].
12.
Memon, R. A.,
C. Grunfeld,
A. Moser,
and
K. R. Feingold.
Tumor necrosis factor mediates the effects of endotoxin on cholesterol and triglyceride metabolism in mice.
Endocrinology
132:
2246-2253,
1993[Abstract].
13.
Nolan, J. P.,
and
M. V. Ali.
Endotoxin and the liver. I. Toxicity in rats with choline deficient fatty livers.
Proc. Soc. Exp. Biol. Med.
129:
29-31,
1968.
14.
Okusawa, S.,
J. A. Gefland,
T. Ikejima,
R. J. Connolly,
and
C. A. Dinarello.
Interleukin-1 induces a shock-like state in rabbits: synergism with tumor necrosis factor and the effect of cyclooxygenase inhibition.
J. Clin. Invest.
81:
1162-1172,
1988[Medline].
15.
Parrillo, J. E.
Pathogenetic mechanisms of septic shock.
N. Engl. J. Med.
328:
1471-1477,
1993[Free Full Text].
16.
Pertoft, H.,
and
B. Smedsrod.
Separation and characterization of liver cells.
In: Cell Separation: Methods and Selected Applications, edited by T. G. Pretlow II,
and T. P. Pretlow. New York: Academic, 1987, p. 1-24.
17.
Savier, E., S. I. Shedlofsky, A. T. Swim,
J. J. Lemasters, and R. G. Thurman. The
calcium channel blocker nisoldipine minimizes the release of tumor
necrosis factor and interleukin-6 following rat liver transplantation.
Transpl. Int. 5 Suppl.:
S398-S402, 1992.
18.
Tracey, K. J.,
B. Beutler,
S. F. Lowry,
J. Merryweather,
S. Wolpe,
I. W. Milsark,
R. J. Hariri,
T. J. Fahey,
A. Zentella,
J. D. Albert,
G. T. Shires,
and
A. Cerami.
Shock and tissue injury induced by recombinant human cachectin.
Science
234:
470-474,
1986[Medline].
19.
Tracey, K. J.,
F. Yuman,
and
D. G. Hesse.
Anti-cachectin/TNF monoclonal antibodies prevent septic shock during lethal bacteremia.
Nature
330:
662-664,
1987[Medline].
20.
Waage, A.,
P. Brandtzaeg,
T. Espevik,
and
A. Halstensen.
Current understanding of the pathogenesis of gram negative shock.
Infect. Dis. Clin. North Am.
5:
781-791,
1991[Medline].
21.
Wall, C. A.,
N. Enomoto,
and
R. G. Thurman.
A choline-rich diet improves survival in a rat model of endotoxin shock (Abstract).
Pharmacologist
39:
48,
1997.
22.
Watanabe, N.,
J. Suzuki,
and
Y. Kobayashi.
Role of calcium in tumor necrosis factor-
produced by activated macrophages.
J. Biochem. (Tokyo)
120:
1190-1195,
1996[Abstract].
23.
Watson, R. W.,
H. P. Redmond,
and
D. Bouchier-Hayes.
Role of endotoxin in mononuclear phagocyte-mediated inflammatory responses.
J. Leukoc. Biol.
56:
95-103,
1994[Abstract].
24.
Wheeler, M. D.,
R. F. Stachlewitz,
and
R. G. Thurman.
Glycine blunts alveolar macrophage activation by a mechanism involving a glycine-gated chloride channel (Abstract).
Toxicology
37:
346,
1998.
25.
Wisse, E.
Ultrastructure and function of Kupffer cells and other sinusoidal cells in the liver.
In: Kupffer Cells and Other Liver Sinusoidal Cells, edited by E. Wisse,
and D. L. Knook. Amsterdam: Elsevier/North-Holland Biomedical, 1977, p. 33-60.
26.
Yamamoto, H.,
K. Hanada,
and
M. Nishijima.
Involvement of diacylglycerol production in activation of nuclear factor
B by a CD14-mediated lipopolysaccharide stimulus.
Biochem. J.
325:
223-228,
1997[Medline].
Am J Physiol Gastroint Liver Physiol 275(4):G862-G867
0002-9513/98 $5.00
Copyright © 1998 the American Physiological Society