Regulation of endothelin synthesis in hepatic endothelial
cells
Ann T.
Eakes and
Merle S.
Olson
Department of Biochemistry, University of Texas Health Science
Center, San Antonio, Texas 78284-7760
 |
ABSTRACT |
Endothelin (ET)
stimulates vasoconstriction and glucose production and mediator
synthesis in the liver. Only hepatic endothelial cells express ET-1
mRNA, and during endotoxemia in the intact rat, a ninefold increase in
hepatic ET-1 mRNA occurs within 3 h of lipopolysaccharide (LPS)
infusion [A. T. Eakes, K. M. Howard, J. E. Miller, and M. S. Olson. Am. J. Physiol. 272 (Gastrointest. Liver
Physiol. 35): G605-G611, 1997]. The present study
defines the mechanism by which hepatic ET production is enhanced during endotoxin exposure. Culture media conditioned by exposure to
endotoxin-treated Kupffer cells stimulated a twofold increase in
immunoreactive ET-1 (irET-1) secretion by liver endothelial cells.
Transforming growth factor-
(TGF-
), tumor necrosis factor-
(TNF-
), LPS, and platelet-activating factor (PAF) were tested for
their ability to stimulate cultured liver endothelial cells to secrete
irET-1. Although TNF-
, LPS, and PAF had no significant effect on
ET-1 synthesis, TGF-
increased ET-1 mRNA expression and irET-1
secretion. In coculture experiments, treating Kupffer cells with
endotoxin caused a doubling of the ET-1 mRNA level in the liver
endothelial cells. This increase in ET-1 mRNA was attenuated by a
TGF-
-neutralizing antibody. Hence, a paracrine signaling mechanism
operates between Kupffer cells that release TGF-
on endotoxin
challenge and hepatic endothelial cells in which TGF-
stimulates
ET-1 mRNA expression and ET-1 secretion; this intercellular signaling
relationship is an important component in the hepatic responses to
endotoxin exposure.
liver; Kupffer cell; lipopolysaccharide; transforming growth
factor
 |
INTRODUCTION |
ENDOTHELIN WAS DESCRIBED as a vasoactive factor found
in conditioned media from cultured vascular endothelial cells by
Yanagisawa et al. (57) in 1988. In fact endothelin (ET) is
accepted as the most potent vasoactive peptide known, producing strong
vasoconstrictor responses in most vascular beds (for review see Ref.
50). ET and its receptors have been identified in many
different tissues, including heart, brain, kidney, lung, liver, spleen,
pancreas, stomach, uterus, testis, and bone (47, 53), as well as in the
constituent cells of the vascular system. The tissue distribution of ET
is nearly identical to that of its receptor(s), indicating the
potential importance of paracrine or autocrine signaling pathways (19).
Studies from our laboratory have shown that in the intact perfused
liver, ET elicits sustained vasoconstriction and an increased hepatic
glucose output (15) and that hepatocytes (14), Kupffer cells (51), and
liver endothelial cells (9) possess functional receptors for ET. In
both hepatocytes (14) and Kupffer cells, ET activates the phospholipase
C signaling pathway. Other workers have shown that in cultured Ito
cells, ET-1 binds to ETA and
ETB receptors, causing contraction
of cells attached to a collagen matrix (23). Additionally, the
contraction of liver sinusoids in vivo after infusion of ET has been
localized to the Ito cells lining the sinusoids (58).
Under normal physiological conditions the low basal levels of ET found
in the circulation are likely not sufficient to activate cellular
processes (36). However, in conditions such as renal failure,
congestive heart failure, and endotoxic shock the level of circulating
ET has been shown to increase significantly (35). During sepsis the
liver is a key organ in the pathophysiological response to endotoxin
[lipopolysaccharide (LPS)]; early liver dysfunction is indicated by
abnormal release of hepatic enzymes into the circulation, and prolonged
exposure to LPS leads to liver failure (21). The first report that
plasma ET levels increase in response to LPS (52) has been confirmed in
different models of endotoxin exposure; these reports (10, 30, 42)
confirm also that the baseline plasma immunoreactive ET-1 (irET-1)
level is quite low. In pigs, a 2-h infusion of LPS caused a significant elevation in irET-1 both in the hepatic portal vein and systemically; this elevation persisted for several hours after termination of the
infusion (40). We have shown recently that a large increase in the
hepatic expression of ET-1 occurs during periods of endotoxin exposure
in the rat and that liver endothelial cells are the primary cell type
involved in this increased expression (10). Endothelial cells in
culture are generally accepted to increase ET production in direct
response to LPS. For example, calf pulmonary artery endothelial cells
increase ET-1 production in response to treatment with as little as 10 ng/ml LPS (42), and microvascular pulmonary endothelial cells also
respond to treatment with LPS by increasing ET-1 mRNA levels within 1 h
of exposure (17). The current study was designed to identify the
mechanism by which LPS exposure caused increased hepatic ET synthesis.
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MATERIALS AND METHODS |
Animals.
Male Sprague-Dawley rats (SASCO, Omaha, NE) weighing between 225 and
300 g were used as a source of primary cultured cells for these
studies. The rats were fed a standard rat chow and water ad libitum and
were handled in compliance with the Animal Welfare Act and according to
the guidelines set forth by United States Department of Agriculture.
All protocols were approved by the Institutional Animal Care and Use
Committee of The University of Texas Health Science Center at San
Antonio, which is accredited by the American Association for the
Accreditation of Laboratory Animal Care.
Reagents.
Collagenase (type IV from Clostridium
histolyticum), protease E (type XIV from
Streptomyces griseus), and BSA
(fraction V and essentially fatty acid free) were purchased from Sigma
(St. Louis, MO). Metrizamide
(2-[3-acetamido-5-N-methylacetamido-2,4,6-triiodobenzamido]-2-deoxy-D-glucose) was purchased from Nyegaard (Oslo, Norway). RPMI 1640 tissue culture medium was purchased from GIBCO (Grand Island, NY). Iron-supplemented calf serum and FCS were purchased from Hyclone (Logan, UT). ET-1, anti-ET-1 antiserum, normal rabbit serum, and goat anti-rabbit IgG were
purchased from Peninsula Laboratories (Belmont, CA). 125I-ET-1 (2,000 Ci/mmol) was
purchased from Du Pont-NEN (Boston, MA). Transforming growth factor-
(TGF-
), tumor necrosis factor-
(TNF-
), and
TGF-
-neutralizing antibody were obtained from R&D Systems
(Minneapolis, MN). TGF-
1 Emax immunoassay kit was
purchased from Promega (Madison, WI). All other reagents
used were of the highest quality commercially available.
Isolation and culture of liver cells.
Rat sinusoidal endothelial cells and Kupffer cells were isolated as
described previously by Knook and Sleyster (26). Kupffer cells (2 × 106 cells/ml) were plated
at 4 ml/60-mm dish or 2 ml/35-mm well, using RPMI 1640 containing 10%
FCS and 5,000 U/ml penicillin and 5,000 µg/ml streptomycin, and
placed in an incubator at 37°C in an atmosphere of 95% air-5%
CO2. The media were changed after 24 h in culture, and the cells were used on the third day in culture. After two rinses with serum-free RPMI containing 0.1% BSA, Kupffer cells were cultured in low-serum RPMI (containing only 2% FCS) during
experiments.
Liver endothelial cells were suspended in RPMI 1640 media containing
0.01% heparin, 2 mM
L-glutamine, and 5,000 U/ml
penicillin and 5,000 µg/ml streptomycin (RPMI) supplemented with 20%
iron-supplemented calf serum. Aliquots of the cell suspension (2 × 106 cells/ml) were plated
at 4 ml/60-mm dish or 2 ml/35-mm well, on dishes coated previously with
rat tail collagen (UBI, Lake Placid, NY), which were incubated at
37°C in an atmosphere of 95% air-5%
CO2. After up to 4 h had been
allowed for attachment, the cells were placed in serum-free medium
containing 0.1% BSA, and other additions were made as indicated in the
various figure legends. Plating efficiency (75-80%) was
determined by counting adherent cells, using a phase-contrast
microscope after placing the cells in serum-free medium. More than 95%
of cultured Kupffer cells and less than 5% of cultured liver
endothelial cells showed positive staining for peroxidase.
Functionality of liver endothelial cells was confirmed by their ability
to internalize acetylated low-density lipoprotein, which was labeled
with the fluorescent tag DiI. Within the rat liver, endothelial cells
are known to be the predominant site of internalization of acetylated
low-density lipoprotein (41).
Coculture of Kupffer cells and liver endothelial cells.
Kupffer cells were plated on Transwell inserts contained within a
six-well plate and maintained in standard culture as described for 3 days before coculture with liver endothelial cells. The liver
endothelial cells were plated onto the surface of a standard six-well
plate and allowed to attach for 2-4 h as described previously. One
hour before coculture the Kupffer cells were placed in RPMI containing
2% fetal bovine serum with or without 50 ng/ml LPS. Immediately before
coculture, liver endothelial cells were placed in RPMI medium
containing 0.1% BSA. The 6-h coculture interval was initiated by
transferring the inserts containing the Kupffer cells into the wells
containing liver endothelial cells.
Northern analyses.
Total RNA samples were prepared from cultured liver endothelial cells
or Kupffer cells using the method of Chomczynski and Sacchi (5).
Northern blot analyses were performed on total RNA samples using a cDNA
either to rat preproendothelin-1, kindly provided by Dr. M. Yanagisawa
(47), or to rat TGF-
1, a generous gift of Dr. Lynda Bonewald (7).
RNA was separated on 1% formaldehyde/agarose gels, transferred to
nylon membranes, and subsequently hybridized with cDNA, which was
random-primer labeled using
[
-32P]dCTP.
Stringency washes were performed sequentially as follows: at room
temperature for 20 min in 2× saline-sodium citrate (SSC) with 1%
SDS, 30 min at 60°C, also in 2× SSC with 1% SDS, 30 min at
60°C in 1× SSC with 0.5% SDS, and 30 min at 50°C in
0.1× SSC with 0.05% SDS. Differences in the amounts of mRNA were
quantitated using a Molecular Dynamics PhosphorImager, and variation in
sample loading was adjusted relative to the level of sample
hybridization to an 18S RNA probe.
RIA of ET-1.
The level of irET-1 was measured in media obtained from cultured liver
endothelial cells. After centrifugation to remove debris from the
culture media, the media were extracted using a
C18 column to reduce the effects
of proteins on the assay, using a modification of the technique
described by Cernacek and Stewart (4). The RIA was adapted from a
commercial protocol and was sensitive over the range of 1-128 pg
irET-1/ml. The samples were incubated overnight in assay buffer
[0.1 M sodium phosphate (pH 7.4), 0.05 M sodium chloride, 0.1%
BSA, 0.01% sodium azide, and 0.1% Triton X-100] with an
anti-ET-1 antibody. According to the manufacturer, the antibody used in
the RIA has only minimal cross-reactivity with big endothelin (<15%)
and ET-3 (<5%). A second overnight incubation was performed with
125I-ET-1. The immune complex was
precipitated with goat anti-rabbit IgG and normal rabbit serum, and the
pellet was collected by centrifugation and counted using a
gamma-scintillation counter.
TGF-
1 assay.
Kupffer cells were plated and treated with LPS for 6 h as described
previously. Media were removed from the cells and stored at
70°C until use, at which time they were acid-activated by adjusting the pH to 2 using 1 N HCl, and then returned to pH 7.4 using
1 N NaOH. Samples were diluted 1:2, 1:4, and 1:8 and subjected to
analysis using a commercially available TGF-
1 ELISA kit according to
the protocol supplied by the manufacturer.
Statistics.
Results are expressed as means ± SE. Data were analyzed for
significance using ANOVA and Newman-Keuls tests with
P < 0.05 considered significant.
 |
RESULTS |
Effect of LPS treatment on ET-1 secretion.
Hepatic endothelial cells were established in culture as described and
then incubated in RPMI plus 10% iron-supplemented calf serum
containing LPS at concentrations ranging from 10 ng/ml to 1 mg/ml for
24 h. At the end of the incubation period the media were removed and
the level of irET-1 was measured by RIA. There was no significant
elevation in the amount of irET-1 released into the culture media at
any LPS concentration tested (Fig. 1). The
highest LPS concentration (1 mg/ml) resulted in decreased levels of
irET-1 in the culture media (56% of the control value), which is
consistent with a toxic effect of LPS at this concentration. This
finding suggested that acute effects of LPS on ET-1 production by the
liver endothelial cells occurred through an indirect mechanism.

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Fig. 1.
Effect of lipopolysaccharide (LPS) treatment on endothelin (ET)-1
secretion: concentration dependence. Liver endothelial cells were
stimulated with indicated concentrations of LPS for 24 h. Culture media
were collected and assayed for immunoreactive ET-1 (irET-1). Each point
is mean ± SE of triplicate determinations from 3 separate
experiments.
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LPS stimulation of TGF-
synthesis by Kupffer cells.
Cultured Kupffer cells were stimulated with LPS, and total RNA was
isolated to assess the change in TGF-
1 mRNA levels. Exposure to LPS
for 6 h increased the level of TGF-
mRNA by 53% relative to control
cells (Fig. 2,
A and
B). The culture media taken from Kupffer cells treated with LPS contained 2.78 ± 0.22 ng/ml
TGF-
1, and the media samples from untreated Kupffer cells contained
1.78 ± 0.15 ng/ml TGF-
1 (Fig. 3).
These data confirm the ability of short-term cultured Kupffer cells to
increase expression and secretion of TGF-
1 in response to LPS
treatment.

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Fig. 2.
LPS stimulation of transforming growth factor- 1 (TGF- 1) mRNA from
Kupffer cells. Kupffer cells were treated with 50 ng/ml LPS for 6 h in
RPMI 1640 medium containing 2% fetal bovine serum. Total RNA (4 µg)
from each Kupffer cell sample was analyzed by Northern analysis with
cDNA probe to TGF- 1. A:
representative Northern blot. Upper band is TGF- mRNA and lower band
is 18S RNA. B: data are expressed as
ratio of treated sample to control value. Each point is mean ± SE
of samples from 5 separate experiments.
* P < 0.003 vs. control.
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Fig. 3.
LPS stimulation of TGF- 1 release from Kupffer cells. Kupffer cells
or media controls were treated with 10 ng/ml LPS for 6 h in RPMI 1640 containing 2% fetal bovine serum. Conditioned media obtained from
Kupffer cells were frozen until assay. Media were thawed and assayed
for presence of TGF- 1 using an ELISA assay kit; media were diluted
1:4 with sample buffer provided. Each point is mean ± SE of
triplicate determinations. * P < 0.001 for conditioned media samples +LPS compared with conditioned
media samples LPS.
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Stimulation of ET-1 secretion by Kupffer cell-conditioned media.
Culture medium (RPMI medium with 10% fetal bovine serum) containing 50 ng/ml LPS or vehicle was incubated for 24 h with cultured Kupffer cells
(conditioned medium) or in empty culture plates (control medium). These
media then were added immediately to liver endothelial cells, which had
been allowed to equilibrate for 2-3 h after plating. After the
liver endothelial cells were incubated for an additional 24 h, the
media were removed and the level of irET-1 was determined by RIA.
Incubation of the liver endothelial cells with medium conditioned by
LPS-treated Kupffer cells resulted in a 60% increase in the level of
irET-1 (Fig. 4) relative to cells treated
with control media without LPS. Neither control media containing LPS
nor conditioned media without LPS resulted in a statistically
significant increase in the amount of irET-1 detected. Therefore, the
capacity of LPS to increase ET-1 synthesis and release from liver
endothelial cells likely occurs via a paracrine signaling mechanism
involving increased cytokine synthesis by the Kupffer cells.

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Fig. 4.
Stimulation of ET-1 secretion by Kupffer cell-conditioned media. RPMI
1640 media with 10% FCS, with or without 50 ng/ml LPS, was conditioned
by exposure to established primary cultures of Kupffer cells for 24 h.
Control media were incubated for 24 h in plates containing no Kupffer
cells. Liver endothelial cells were stimulated with media for 24 h.
Culture media were collected and assayed for irET-1. Data are expressed
relative to mean value of the control media for each experiment. Each
point is mean ± SE of 5 determinations.
* P < 0.0005 vs. control media
samples and <0.001 vs. Kupffer cell-conditioned media without LPS.
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Effect of various agonists on ET-1 secretion.
Because Kupffer cells stimulated with LPS have been shown to increase
production of different mediators (38, 45), liver endothelial cells
were treated with various agonists that are associated with endotoxin
challenge of the liver. After incubation for a 24-h period, the culture
media were collected to determine the amount of irET-1 released by the
endothelial cells. Table 1 shows that 100 pM TGF-
resulted in greater than a 2.5-fold increase in irET-1,
whereas the other agonists tested, TNF-
, platelet-activating factor
(PAF), and LPS, failed to cause a statistically significant increase in
the irET-1 secreted relative to controls. This lack of significant
increase in irET-1 was observed at concentrations of agonist spanning
several orders of magnitude and several different incubation intervals
(data not shown).
TGF-
stimulation of ET-1 synthesis.
TGF-
(100 pM) was added to liver endothelial cells 2-3 h after
plating, and total RNA was collected at the time of addition as well as
4, 6, 8, 12, and 18 h later. The level of ET-1 mRNA was assessed by
Northern analysis with a cDNA probe for ET-1. Elevation of ET-1 mRNA
was detected as early as 4 h after addition of TGF-
(Fig.
5, A and
B). The maximal level of ET-1 mRNA,
roughly fivefold greater than control, was achieved after 8 h of
stimulation with 100 pM TGF-
, with a subsequent decline over the
next 10 h. The concentration dependence of TGF-
stimulation of ET-1
mRNA expression also was determined. Liver endothelial cells were
treated with vehicle or increasing concentrations of TGF-
for 8 h,
at which time total RNA was collected and the levels of mRNA for ET-1
were subsequently assessed by Northern analysis using a cDNA probe for
ET-1. An elevation in ET-1 mRNA was first noted at 10 pM TGF-
, and
the increase appeared to be maximal at 100 pM (Fig. 6, A and
B).

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Fig. 5.
TGF- stimulation of ET-1 mRNA: time dependence. Liver endothelial
cells were stimulated with 100 pM TGF- for indicated times. Total
RNA (10 µg) from each time point was analyzed by Northern analysis
with cDNA probe to ET-1. A:
representative Northern blot. Upper band is ET-1 mRNA and lower band is
18S RNA. B: data are expressed as
ratio of treated sample to control value. Each point is mean ± SE
of 3 separate experiments. * P < 0.05 vs. untreated control.
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Fig. 6.
TGF- stimulation of ET-1 mRNA: concentration dependence. Liver
endothelial cells were stimulated with indicated concentrations of
TGF- for 8 h. Total RNA (10 µg) from each dose was analyzed by
Northern analysis with cDNA probe to ET-1.
A: representative Northern blot. Upper
band is ET-1 mRNA and lower band is 18S RNA.
B: data are expressed as ratio of
treated sample to untreated control. Each point is mean ± SE of 3 separate experiments. * P < 0.03 vs. control. ** P < 0.005 vs. control.
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TGF-
stimulated the secretion of irET-1 from liver endothelial cells
in a concentration-dependent manner, which was maximal at 100 pM, 6.4 ± 1.1 pg/106 cells (Fig.
7). A marked increase in the
secretion of ET-1 occurred after 24 h in response to as little as 25 pM
TGF-
(3.2 pg/106 cells compared
with 2.4 pg/106 cells in the
control). The maximal level of irET-1 was that measured at 24 h of
exposure. However, even after 12 h of treatment with TGF-
a
detectable difference in the amount of irET-1 released into the culture
medium was apparent (data not shown). This lag in irET-1 synthesis
after stimulation has been observed in porcine aortic endothelial cells
(29). Taken together, the present results clearly demonstrate the
ability of TGF-
to stimulate the production of ET-1 in liver
endothelial cells and implicate a paracrine signaling mechanism
operating between hepatic sinusoidal cells. Involved in this
intercellular signaling mechanism, LPS-treated Kupffer cells release
the cytokine TGF-
, which stimulates ET-1 synthesis in the
endothelial cell.

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Fig. 7.
TGF- stimulation of ET-1 secretion: concentration dependence. Liver
endothelial cells were stimulated with indicated concentrations of
TGF- for 24 h. Culture media were collected and assayed for irET-1.
Each point is mean ± SE of 3 determinations from 4 separate
experiments. * P < 0.002 vs.
control.
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TGF-
-neutralizing antibody inhibits ET-1 production
by liver endothelial cells cocultured with LPS-stimulated Kupffer
cells.
Liver endothelial cells cocultured with LPS-treated Kupffer cells
contained approximately twofold more ET-1 mRNA than cells cocultured
with untreated Kupffer cells (Fig. 8,
A and
B). When a panspecific neutralizing
antibody to TGF-
was included in the medium the increase in ET-1
mRNA was diminished, indicating that a substantial portion of the
LPS-mediated Kupffer cell stimulation of ET-1 synthesis by liver
endothelial cells was a consequence of TGF-
generation by the
Kupffer cells. The amount of neutralizing antibody was in
sufficient excess to have completely blocked any effect of the
TGF-
1 detected in the Kupffer cell medium (2.7 ng/ml),and a 10-fold
increase in the amount of antibody used resulted in no further
diminution in the level of ET-1 mRNA (data not shown). The effect of
coculture on the release of irET-1 was not measured because media
collected after only 6 h in culture would have accumulated insufficient
irET-1 for differences in synthesis and release to be detected.
Additionally, we anticipated that binding of released ET to Kupffer
cells during the coculture interval would further decrease measurable
irET-1.

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Fig. 8.
TGF- neutralizing antibody inhibits increased ET-1 production
stimulated by coculture of liver endothelial cells with LPS-stimulated
Kupffer cells. Kupffer cells were pretreated with 50 ng/ml LPS for 1 h
in RPMI 1640 medium containing 2% fetal bovine serum. Immediately
before coculture, indicated liver endothelial cell samples were treated
with 10 µg/ml of panspecific neutralizing antibody to TGF- . After
pretreatment, Kupffer cells were placed in coculture with liver
endothelial cells for additional 6 h. Total RNA (7.5 µg) from each
liver endothelial cell sample was analyzed by Northern analysis with
cDNA probe to ET-1. A: representative
Northern blot. Upper band is ET-1 mRNA and lower band is 18S RNA.
B: data are expressed as ratio of
treated sample to control value. Each point is mean ± SE of 4 separate experiments. * P < 0.0002 vs. Kupffer cell control.
** P < 0.001 vs. LPS-treated
Kupffer cell and P < 0.02 vs.
Kupffer cell control.
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 |
DISCUSSION |
In pathophysiological episodes such as sepsis,
ischemia-reperfusion injury, renal failure, and congestive
heart failure the plasma levels of ET-1 are elevated, reflecting
presumptive increases in ET-1 synthesis (2, 10, 18, 32). It is
generally accepted that ET itself is not stored within the cell and
that increased levels of peptide secretion usually are accompanied by
elevated levels of ET mRNA. However, it has been suggested recently
(46) that low LPS concentrations might promote ET-1 release by a
posttranscriptional mechanism located upstream of big ET-1, without
altering the level of ET message. Therefore, in the present study we
have measured both the release of irET-1 and the levels of mRNA for
preproendothelin-1.
Production of ET or TGF-
in response to LPS.
Tissue-specific differences in mRNA for ET-1 in rat models of sepsis
have been noted, with heart and lung (24) and liver (10) exhibiting an
elevation of ET-1 mRNA, whereas kidney or skeletal muscle shows little
change (24). Endothelial cells from nonhepatic tissues show increased
ET-1 production in direct response to LPS (17, 42). However, treatment
of cultured liver endothelial cells with LPS did not significantly
increase either irET-1 detected in the culture media or cellular ET-1
mRNA (data not shown). These observations led to the hypothesis that
LPS action must involve an intermediate agonist possibly acting through another cell type in the liver. Kupffer cells, the resident macrophage of the liver, are colocalized with endothelial cells within the sinusoid and are the primary hepatic site of LPS sequestration (55).
Cultured rat Kupffer cells metabolically process LPS (12), and the
ability of the Kupffer cell to respond to LPS has been well documented
(21). Moreover, in the intact LPS-treated rat, Kupffer cells are
thought to modulate both the functional and ultrastructural properties
of sinusoidal endothelial cells (48).
The production and significance of TGF-
during long-term hepatic
changes (e.g., fibrosis and regeneration) have been extensively investigated. However, there is a surprising paucity of data regarding the acute hepatic production of TGF-
in response to LPS. Cultured rat Kupffer cells are known to produce increased amounts of TGF-
in
response to LPS or to bacterial cell wall preparations (31); in these
experiments 3.5 × 106 mixed
sinusoidal cells were plated and the Kupffer cell population was
selected by their rapid adherence to the culture surface [
1.2 × 106 Kupffer cells
(54)]. Exposure to 100 ng/ml LPS for 24 h yielded 2.5 ng active
TGF-
in the medium. Unstimulated cells secreted only 0.18 ng
TGF-
, so the increase in this period was 1.9 ng active
TGF-
/106 cells. Our experiments
used 4 × 106 Kupffer cells
stimulated with 10 ng/ml LPS for a shorter (6 h) stimulation period,
and we obtained an increase of 1 ng/ml TGF-
(Fig. 3); this
represents an increase of 0.25 ng
TGF-
/106 cells. Taken together,
these data suggest that within 6 h of LPS exposure Kupffer cells
secrete sufficient TGF-
to cause increased expression of
preproendothelin-1 mRNA in liver endothelial cells (1 ng/ml TGF-
= 40 pM; see Fig. 6B) and that
continuing secretion of TGF-
readily yields enough mediator (2.5 ng/ml TGF-
= 100 pM) to cause the observed increase in ET-1
production by liver endothelial cells (see Fig. 7). These effects are
directly confirmed by our coculture data (Fig. 8).
Hepatic production of TGF-
: isoforms and activation
state.
In cells from normal rat liver, Kupffer cells express more TGF-
1
mRNA than any other cell type (1, 8). Although signals for TGF-
2 and
TGF-
3 are much weaker than for TGF-
1, Kupffer cells also express
more mRNA for TGF-
2 than any other cell type (1, 8) and express as
much TGF-
3 mRNA as Ito cells (1). In the present study TGF-
1
levels were elevated in Kupffer cells when exposed to the same
concentration of LPS used by us to achieve maximal increases in the
synthesis of the lipid mediator PAF (59) and the expression of the
inducible form of nitric oxide synthase (39). The neutralizing antibody
used in the present coculture experiments does not distinguish between
the different isoforms of TGF-
; it is possible that Kupffer cells
additionally synthesize and release elevated levels of TGF-
2 or
TGF-
3 in response to LPS. Further neutralization studies are
necessary, using antibodies specific for other isoforms of TGF-
and
for other cytokine(s) (e.g., interleukins 1 and 6) known to be released
by the Kupffer cell during endotoxic episodes (45).
Unstimulated Ito cells secrete TGF-
1 (33), so it is possible that
these cells may also contribute to the level of ET synthesis and
release by liver endothelial cells. Two factors influencing such
effects remain to be clarified. First, it is not clear how much LPS
passes the Kupffer cells and sinusoidal endothelial cells to reach the
Ito cells. Second, to our knowledge there is no report that LPS
increases TGF-
secretion by Ito cells.
TGF-
and TNF-
as secretagogues for
ET.
TGF-
was first reported to increase both the expression of mRNA for
preproendothelin in, and the secretion of ET from, porcine aortic
endothelial cells (29). Since then, exogenously added TGF-
has been
confirmed as a secretagogue for ET in a number of different cell types,
most recently in astrocytes (56) and human decidualized endometrial
cells (28). Antibodies or antisense oligodeoxynucleotides against
TGF-
can ablate the effects of endogenous TGF-
, suggesting that
autocrine production of the cytokine can also modulate ET production
(49). Pharmacological studies in the intact rat are consistent with the
concept that TGF-
1 stimulates the synthesis and release of ET (16).
The secretagogue control mechanism has yet to be fully characterized; in the MDCK cell line treatment with TGF-
increases the half-life of
preproendothelin mRNA, suggesting that this is one mechanism for
increased ET secretion (22). It has been suggested also that TGF-
may directly stimulate expression of the gene for ET-1 (27). TNF-
activates ET-1 synthesis in human amnion cells (3) and in bovine
endothelial cells of aortic (34) and cerebral (11) origin. The lack of
a significant increase in ET-1 synthesis with TNF-
treatment again
distinguishes the liver endothelial cell from endothelial cells derived
from other tissues.
TGF-
effects on ET binding.
The binding of ET to its receptors is rapid and essentially
irreversible, which means that substantial downregulation of ET receptors occurs when peptide levels are elevated; this downregulation can be autologous (13). Although endotoxemia elevates systemic ET
levels (see above), treatment of rats with LPS enhances rather than
decreases the contractile responses of the hepatic portal vein to ET
(43). This suggests that in the intact animal LPS itself or an evoked
cytokine might directly upregulate ET receptors. Treatment with TGF-
has been reported both to increase binding of ET-1 in cultured rabbit
costal chondrocytes (25) and to decrease the maximal binding for ET-1
binding [in A617 cells, a vascular smooth muscle-derived cell
line (6)]. We have detected no substantial change in the binding
of ET-1 to liver endothelial cells as a result of TGF-
treatment
(9).
Intercellular interactions involving TGF-
and ET.
These interactions have been investigated in cardiovascular models. The
ability of platelets to stimulate the production of ET-1 by vascular
endothelial cells is thought to be mediated predominantly by platelet
release of TGF-
(37). Moreover, TGF-
causes cardiac hypertrophy
in vivo but not directly in cultured cardiac myocytes; in coculture
TGF-
stimulates nonmyocyte cardiac cells to secrete ET (20), which
is directly hypertrophic. To our knowledge, there is only one other
report investigating comparable hepatic interactions. Rieder et al.
(44) showed that ET production from guinea pig sinusoidal endothelial
cells is elevated both by TGF-
and by conditioned medium from
LPS-stimulated Kupffer cells (maximal increase ~50% in both cases).
We have extended these findings by measuring ET gene expression in
addition to ET production, by obtaining a greater (2-fold) increase in
ET production using coculture experiments, and by using antibodies to
confirm that TGF-
is a major effector produced by Kupffer cells.
The present studies suggest a model of paracrine signaling to describe
the mechanism for the elevation of hepatic ET-1 levels after endotoxin
exposure. LPS released into the portal circulation challenges the liver
where it is bound and cleared by Kupffer cells, resulting in the
production of several inflammatory mediators, including the cytokine
TGF-
. Mediators released by Kupffer cells bind to receptors on liver
endothelial cells and instigate an increase in the expression and
release of ET-1. ET-1 synthesized in this fashion may exhibit actions
in various cell types of the liver, causing contraction of the hepatic
sinusoids, increased glycogenolysis in hepatocytes, and further
synthesis of inflammatory mediators.
 |
ACKNOWLEDGEMENTS |
The authors acknowledge the advice and assistance of Lynda Bonewald
and Jennifer Rosser in assessment of TGF-
1 levels, Stephen A. K. Harvey for helpful and insightful discussions, and the skillful technical assistance of Lynnette Walters and Michael DeBuysere.
 |
FOOTNOTES |
This work was supported by the National Institute of Diabetes and
Digestive and Kidney Diseases Grant DK-19473. A. T. Eakes was a Robert
A. Welch Foundation predoctoral fellow.
Address for reprint requests: M. Olson, Biochemistry Dept., Univ. of
Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San
Antonio, TX 78284-7760.
Received 20 October 1997; accepted in final form 6 March 1998.
 |
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