Department of Medicine, University of North Carolina, Chapel Hill, North Carolina 27599-7080
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ABSTRACT |
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The hepatic
stellate cell (HSC), after a fibrogenic stimulus, is transformed from a
quiescent to an activated phenotype, including the induction of
responsiveness to a variety of agonists. We investigated the activation
of nuclear factor-B (NF-
B) and the expression of the
NF-
B-responsive genes intercellular adhesion molecule 1 (ICAM-1) and
macrophage inflammatory protein-2 (MIP-2) in freshly isolated and
culture-activated HSC by tumor necrosis factor-
(TNF-
) or
interleukin-1
. Inhibitor-
B was rapidly (<15 min) degraded, and
NF-
B activity was induced in culture-activated but not in freshly
isolated HSC after cytokine stimulation. After 30 min of stimulation,
immunofluorescence revealed that the NF-
B p65 subunit was
predominantly found in the nuclei of activated HSC compared with the
cytoplasmic localization in unstimulated cells. No nuclear
translocation appeared in freshly isolated HSC after stimulation,
despite the presence of functional TNF-
receptors. NF-
B nuclear
translocation appeared first partially after 4-5 days and
completely after 9 days in culture. Consistent with this time course
TNF-
induced the mRNA of the NF-
B-dependent genes ICAM-1 and
MIP-2 in activated but not in quiescent HSC. Therefore, cytokines
induce NF-
B activity and ICAM-1 and MIP-2 mRNAs in activated but not
in quiescent HSC, through a postreceptor mechanism of regulation.
tumor necrosis factor-; nuclear factor-
B; interleukin-1; intercellular adhesion molecule 1
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INTRODUCTION |
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HEPATIC FIBROSIS is characterized by increased
deposition of extracellular matrix proteins within the interstitial
space. The activated hepatic stellate cell (HSC, also called Ito cells, lipocytes, or fat-storing cells) is predominantly responsible for the
synthesis of the extracellular matrix proteins during hepatic
fibrogenesis (15, 50, 67, 73). Fibrotic hepatic injury induces the HSC
to undergo an activation process (16, 21) that includes stimulation of
proliferation, a phenotypic transformation to a myofibroblast-like cell
with the loss of cellular retinol stores, and the appearance of smooth
muscle -actin, increased synthesis of extracellular matrix proteins,
and contractility (42, 53, 56, 65). Many of the morphological and
metabolic changes associated with HSC activation during fibrogenesis in vivo are also observed in HSC grown in culture on plastic (8, 15-17, 26). These changes include the expression of new receptors, such as platelet-derived growth factor-
receptor (77), transforming growth factor-
receptors 1 and 2 (18), and ferritin receptor (57).
Other receptors, such as receptors for insulin-like growth factor I (4)
and endothelin (30, 59), are present on both quiescent and activated
HSC.
Interleukin-1 (IL-1) and tumor necrosis factor- (TNF-
) are
elevated during hepatic inflammation, such as alcoholic liver disease
(47), and contribute to the activation of HSC. TNF-
and IL-1 are
potent inducers of nuclear factor-
B (NF-
B), a key transcription
factor that induces genes involved in inflammation, responses to
infection, and stress (1, 70). DNA binding activity of
NF-
B is demonstrated in activated but not in quiescent HSC (Ref. 40,
and R. A. Rippe, unpublished data), and activation of HSC is associated
with the nuclear translocation of NF-
B (40). Using
differential display, we demonstrated the expression of intercellular
adhesion molecule 1 (ICAM-1) in HSC that were activated in culture or
in vivo, but not in quiescent HSC (26). Because the ICAM-1 gene
contains an NF-
B binding site and its transcription is stimulated by
NF-
B (29, 39, 60), this observation provides functional support for
a critical role of NF-
B in the activation of HSC.
The classic NF-B protein is a heterodimer of p50 (NF-
B-1) and p65
(rel A) subunits, but proteins that constitute the NF-
B family form
a variety of homodimers and heterodimers (13, 70). NF-
B is retained
in an inactive form in the cytoplasm through association with one of
the I-
B inhibitory proteins, such as I-
B-
(24).
After cellular stimulation, inhibitor-
B (I-
B)-
is
phosphorylated and ubiquinated and undergoes proteolysis with the
proteasome complex, enabling NF-
B to translocate into the nucleus,
where it stimulates the transcription of several genes by interacting
with
B binding sites (2).
The effects of TNF- and IL-1 on NF-
B signal transduction in
quiescent and activated HSC are unknown. This study demonstrates that
although quiescent HSC express TNF-R1 and have binding sites for
TNF-
, TNF-
does not induce NF-
B activity. On the other hand,
when activated HSC are treated with TNF-
or IL-1, I-
B-
is
degraded, NF-
B translocates to the nucleus, the DNA binding activity
of NF-
B increases, and NF-
B-responsive genes are induced. Thus
TNF-
and IL-1 induce NF-
B binding activity and subsequent gene
expression in activated but not in quiescent HSC, and this block in
quiescent HSC occurs at a postreceptor step.
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METHODS |
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HSC isolation and culture.
HSC were isolated by sequential digestion of the liver of male
Sprague-Dawley rats with Pronase and collagenase, followed by
arabinogalactan gradient ultracentrifugation (58). The purity of the
isolated HSC as examined by phase contrast- and ultraviolet-exited fluorescence microscopy was 98%, and cell viability as examined by
trypan blue exclusion was 99%. HSC were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and penicillin and/or
streptomycin. After 3 days in culture, the HSC had a quiescent
phenotype, as described previously (20, 68). After 14 days of culture,
our previous studies have documented activation of HSC by loss of retinol droplets, expression of smooth muscle -actin, and expression of collagen
1(I) mRNA levels (26, 58).
RNA extraction and amplification by RT-PCR.
For TNF- stimulation experiments, freshly isolated or
culture-activated HSC were seeded for 3 days on plastic, before
incubation with recombinant murine TNF-
(10 ng/ml; R & D Systems,
Minneapolis, MN) in serum-free medium for 12 h. RNA was isolated from
freshly isolated or culture-activated HSC using the Trizol method
(GIBCO, Grand Island, NY) according to the manufacturer's
specifications.
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TNF-R binding assay.
A competitive ligand binding assay using cold competitive TNF- and
125I-labeled TNF-
was used to
determine surface expression of TNF-R on quiescent and activated HSC.
Freshly isolated HSC and culture-activated HSC were seeded in 24-well
plates in complete medium for 3 days. Cells were washed with RPMI
medium containing 10% FBS before incubating the cells in the same
medium for 3 h at 4°C in the presence of 800 pM human
125I-TNF-
(DuPont NEN, Boston,
MA). Competition studies were carried out with increasing
concentrations of unlabeled human TNF-
(2-80 nM, R & D
Systems). Incubations were terminated by removal of the incubation
medium and five washes with ice-cold RPMI-10% FBS, before lysing the
cells in 0.1% SDS-PBS and measuring the cell-bound radioactivity in a
gamma counter. Experiments were performed in triplicate. Data are
displayed as mean specific binding per microgram of DNA. DNA
concentration was determined by fluorometric assay.
Whole cell extracts.
Freshly isolated or culture-activated HSC were grown in 60-mm dishes,
washed with PBS, and cultured in serum-free medium for 24 h before a
30-min stimulation with TNF- (10 ng/ml) or IL-1
(2.5 ng/ml).
Cells were washed with ice-cold PBS and lysed in Dignam C buffer (420 mM NaCl, 1.5 mM MgCl2, 20 mM
HEPES, pH 7.0, 0.2 mM EDTA, 25% glycerol, and 0.5 mM dithiothreitol),
containing protease and phosphatase inhibitors. Lysates were incubated
on ice for 10 min and subsequently rotated at 4°C for 30 min. Cell membranes were pelleted by centrifugation at 14,000 rpm, and the resulting supernatant was aliquoted and stored at
80°C.
Protein concentration was determined using the Bio-Rad protein assay
(Bio-Rad Laboratories, Hercules, CA).
Electrophoretic mobility shift assay.
Freshly isolated HSC or culture-activated HSC were washed with PBS,
starved in serum-free medium for 24 h, and incubated with TNF- (10 ng/ml) or IL-1
(2.5 ng/ml) for 30 min. Whole cell extracts (5 or 1 µg for time-course experiment in Fig.
5B) were incubated with a
double-stranded radiolabeled oligonucleotide containing a consensus
B site (GGCTGGGGATTCCCCATCT) (25) or the previously described
binding site of footprint 4 (FP4) on the collagen
1(I) promoter (58)
(CGGGAGGGGGGGAGCTGGGTG), separated by electrophoresis, and analyzed by
autoradiography as described previously (33). For antibody
supershifting analysis, nuclear extracts were preincubated with 1 µl
of rel A antibody directed against the COOH-terminus portion of the
molecule (Rockland, Gilbertsville, PA) for 15 min at RT before the
addition of the binding buffer and probe as described (34). The
specificity of the probes was evaluated by incubating the whole cell
extracts with a 100-fold excess of unlabeled oligonucleotide. All
mobility shift experiments were performed in three independent experiments, except for the time course (Fig.
5B), which was performed in two
independent experiments.
p65 Western blots.
Whole cell extracts (10 µg) were electrophoresed on 10%
SDS-polyacrylamide gel and blotted onto nitrocellulose membrane as described previously (26). After 60 min in 10% blocking buffer [10% nonfat milk in 20 mM Tris · HCl, pH 7.6, 137 mM NaCl, 0.1% Tween 20 (TBST)], the membrane was incubated
with either a rabbit anti-p65 antibody (Rockland) or an I-B-
antibody (Santa Cruz Biotechnology, Santa Cruz, CA) diluted 1:1,000 in
5% blocking buffer (5% nonfat milk in TBST) for 45 min. After three
washes with TBST, the membrane was incubated for another 30 min with horseradish peroxidase-conjugated goat anti-rabbit antibody (Amersham, Arlington Heights, IL) diluted 1:1,000 in 5% blocking buffer, washed
in TBST, and then developed using the enhanced chemiluminescence light-detecting kit (Amersham) according to the manufacturer's recommended instructions (33). To ensure protein integrity and equal
loading, 10 µg of total cell extract from both activated and
quiescent HSC were separated on a 10% SDS-PAGE followed by Coomassie
staining as described previously (32).
JNK assay.
Quiescent and activated HSC were stimulated for 30 min with TNF- (10 ng/ml) or IL-1
(2.5 ng/ml), and Jun NH2-terminal kinase (JNK) activity was assessed using an in vitro kinase assay as described
previously. Recombinant glutathione S-transferase (GST)-c-Jun protein
(amino acids 1-79), containing the activation domain of c-Jun, a
mutated GST-c-Jun with Ser63 and
Ser73 mutated to Ala
(GST-c-JunAA), or GST protein alone was utilized as a
substrate. Whole cell extracts (5-10 µg) prepared
from quiescent and activated HSC were incubated with 0.5 µg of
substrate protein linked to glutathione-Sepharose beads. After
extensive washing of the complexes, the kinase reactions were performed
with [
-32P]ATP. The
proteins were fractionated using 12.5% SDS-PAGE and then visualized
and quantitated using PhosphoImager analysis (Molecular Dynamics,
Sunnyvale, CA). Coomassie staining was used to demonstrate equal
protein loading. Data represent the means of three independent experiments (±SD). Representative gels are shown.
p65 Immunofluorescence.
HSC grown on chamber slides (Nunc, Naperville, IL) were stimulated for
30 min with TNF- (10 ng/ml), briefly washed with PBS, and then fixed
with ice-cold 100% methanol for 10 min. After blocking for 30 min with
10% nonimmune goat serum (NGS, Sigma Chemicals, St. Louis, MO) cells
were incubated with rabbit anti-p65 antibody (Rockland) diluted 1:200
in 10% NGS-PBS. Slides were washed three times with PBS before 30-min
incubation with rhodamine isothiocyanate-conjugated goat anti-rabbit
IgG antibody (Jackson ImmunoResearch, West Grove, PA) and diluted 1:100
in 10% NGS-PBS. The p65 localization was visualized with a
fluorescence light microscope.
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RESULTS |
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TNF-R1 and IL-1 receptor mRNA is expressed by quiescent and activated HSC. Because the status of TNF and IL-1 receptors in HSC is unknown, we first wanted to assess the expression of their mRNAs in quiescent and activated HSC. Using RT-PCR, we demonstrated the presence of mRNA for the TNF-R1 and the IL-1R in both quiescent and activated HSC (Fig 1A). For TNF-R mRNA analysis, various primer sets amplified parts of the transmembrane and cytoplasmic domain. This demonstrates the transcription of an intact TNF-R. Because it had been previously shown that Kupffer cells, hepatocytes, and endothelial cells are able to express ICAM-1 (36, 63, 66), we used the same cDNAs in a PCR amplifying ICAM-1 as a negative control. We previously demonstrated that freshly isolated HSC do not express ICAM-1 (26). As expected ICAM-1 mRNA was detected only in the activated HSC, demonstrating the purity of the HSC preparation. It is therefore unlikely that the detected mRNA for the TNF-R1 or IL-1R reflects contamination from other liver cell populations.
|
TNF- binds to quiescent and activated HSC.
To extend our finding of TNF-
R1 mRNA expression in quiescent and
activated HSC, expression of TNF receptors on the cell surface of
quiescent and activated HSC was demonstrated using a competitive ligand
binding assay. Binding of
125I-TNF-
to quiescent and
activated HSC was inhibited by competitive nonradioactive TNF-
in a
dose-dependent way (Fig. 1B). A
100-fold excess of unlabeled TNF-
reduced
125I-TNF-
binding to about
5-10% of total bound
125I-TNF-
to quiescent and
activated HSC, as shown in Fig. 1C.
Thus both quiescent and activated HSC bind TNF-
. Differences in
TNF-R number have generally not been reflected in differences in TNF downstream effects (31, 38).
TNF- and IL-1
increase
NF-
B binding activity in activated but not in quiescent
HSC.
NF-
B binding activity is increased in activated HSC after TNF-
and IL-1
stimulation, as demonstrated by electrophoretic mobility
shift assays (Fig.
2A,
lanes 3 and
4). However, over a range of TNF-
and IL-1
concentrations, these cytokines failed to induce NF-
B
binding activity in quiescent HSC (Fig.
2A, lanes 6 and 7 and data not
shown). The components of the NF-
B complex induced by cytokine
stimulation contains the major transactivator protein p65 as shown by
antibody supershifting (Fig. 2B). To
demonstrate the presence and integrity of the NF-
B protein in whole
cell extracts of quiescent HSC, we performed Western blots for the p65
subunit (Fig. 2C) using the same
whole cell extracts of activated and quiescent HSC as for the
electrophoretic mobility shift assay (EMSA). With 10 µg
whole cell extracts the p65 protein expression was lower in the
quiescent than in the activated HSC but clearly detectable. Coomassie
staining of the same protein extracts from quiescent or activated HSC
used for Western blot or EMSA demonstrate that the difference in p65
expression was not due to unequal protein loading or degradation (Fig.
2D). The difference in protein
pattern expression between quiescent and activated HSC may reflect
their stage of activation. Expression of the p65 protein was the same in untreated and TNF-
- or IL-1
-treated cells. In particular, there was no evidence of NF-
B p65 degradation in quiescent HSC.
|
TNF- and IL-1
induce
I-
B-
degradation in activated but not
in quiescent HSC.
To further investigate the mechanism for the failure of NF-
B
induction after cytokine stimulation in quiescent HSC, we analyzed I-
B-
protein expression in quiescent and activated HSC after TNF-
and IL-1
stimulation by Western blot. I-
B-
degradation has been shown to be a prerequisite for NF-
B activation (27). Both
cytokines induce a rapid degradation of I-
B-
in activated HSC
(Fig 3, A
and B,
right) but not in quiescent HSC
(Fig. 3, A and
B,
left). This result is consistent
with preceding EMSA data, which demonstrated nuclear translocation of
NF-
B (p65) in activated but not in quiescent HSC.
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JNK activity in quiescent and activated HSC after cytokine
stimulation.
Because TNF- and IL-1
stimulate JNK activity in a variety of
cultured cells (37, 74), we assessed the ability of TNF-
and IL-1
to stimulate JNK activity in quiescent and activated HSC. JNK activity
was measured by a solid state in vitro kinase assay. Phosphorylated
GST-c-Jun was visualized after protein fractionation using 12.5%
SDS-PAGE and quantitated using PhosphoImager analysis (Fig.
4). TNF-
induced JNK activity 1.5 ± 0.2-fold in quiescent HSC and 2.4 ± 0.3-fold in activated HSC
(where P < 0.05 for untreated vs.
TNF-
-treated cells for both activated and quiescent HSC). IL-1
stimulation led to a 1.6 ± 0.2-fold and 4.3 ± 1.6-fold increase of JNK activity in quiescent and activated HSC, respectively
(P < 0.05 for untreated vs.
IL-1
-treated cells for both activated and quiescent HSC). A recent
study has also demonstrated that IL-1 and TNF-
stimulate JNK in
activated HSC (55). The relatively lower induction of JNK activity in
quiescent HSC might be due to a higher basal activity after the
isolation procedure. We found a 6.6 ± 3.3-fold higher basal JNK
activity in freshly isolated HSC than in the culture-activated HSC
(data not shown). These data demonstrate that the TNF-
signal
transduction pathway that activates JNK is functional in both quiescent
and activated HSC.
|
TNF- starts to induce NF-
B activity
in cultured HSC 4 days after isolation.
We determined the time point when TNF-
starts to induce the NF-
B
activity during the activation process of HSC in culture by studying
p65 (rel A) subcellular localization and NF-
B binding activity. HSC
up to 3 days in culture show an unchanged cytoplasmic pattern of p65
staining with no appreciable nuclear p65 staining after TNF-
stimulation (Fig
5A, A-F).
On culture day 4, we first detected
nuclear translocation of p65 in about 50% of the cells after TNF-
stimulation (Fig 5A, I), whereas the
other 50% (Fig 5A, H) showed no
change in the p65 staining pattern compared with the unstimulated
control cells (Fig 5A, G), suggesting
high heterogeneity among HSC during the activation process. After 6 and
14 days in culture, nearly all HSC show nuclear translocation of p65
after TNF-
stimulation (Fig 5A, K
and M).
|
TNF- induced ICAM-1 and MIP-2 expression in
activated but not in quiescent HSC.
Finally, we investigated the cytokine-mediated induction of ICAM-1 and
MIP-2 mRNAs by RT-PCR in HSC cultured on plastic for 3 or 14 days (Fig.
6). The expression of the genes for ICAM-1 (29, 39) and MIP-2 (76) is stimulated by NF-
B through their
B
binding sites. TNF-
increased the mRNA expression of ICAM-1 and
MIP-2 in activated HSC. Compared with freshly isolated HSC (Fig.
1A) there is some basal expression
of ICAM-1 mRNA in HSC cultured for 3 days on plastic, indicating
initiation of the activation process. However, consistent with the lack
of NF-
B activation after cytokine stimulation, there is no increased
ICAM-1 or MIP-2 mRNA in 3-day-cultured HSC after TNF-
treatment
(Fig. 6).
-Actin mRNA expression was used to demonstrate equal
amounts of RNA in each PCR. It is unlikely that the ICAM-1 expression
in 3-day-cultured HSC reflects contamination from other liver cells
because Kupffer cells, hepatocytes, and endothelial cells respond to
TNF-
stimulation with increased ICAM-1 expression (12, 19, 49, 54).
|
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DISCUSSION |
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This study focuses on the effects of TNF- on NF-
B signal
transduction in quiescent and activated HSC with corroborative evidence
provided by studies with IL-1
. Although quiescent HSC express TNF-R1
mRNA and have binding sites for TNF-
, this cytokine does not induce
NF-
B activity in these cells. On the other hand, when activated HSC
are treated with TNF-
or IL-1, I-
B-
is rapidly degraded,
NF-
B translocates to the nucleus, the DNA binding activity of
NF-
B increases, and NF-
B-responsive genes (ICAM-1 and MIP-2) are
induced. Thus the TNF-
signal transduction pathway for NF-
B appears to be fully functional in activated HSC but blocked or inhibited at a postreceptor level in quiescent HSC. The presence of
TNF-R on quiescent HSC was investigated by three independent methods.
First we showed the presence of TNF-R mRNA using primers targeting the
transmembrane and cytoplasmic region. Second, ligand binding studies
demonstrated the presence of TNF-R on the surface of quiescent HSC.
Third, a small but significant induction of JNK activity was observed
in TNF-
-treated quiescent HSC. Furthermore, a recent study has
demonstrated that IL-1
induces morphological and biochemical changes
in quiescent HSC (62), providing indirect evidence for the presence of
an IL-1R. Overall, these data support a postreceptor defect of the
NF-
B signaling pathway in quiescent HSC.
Recent studies have greatly increased our understanding of the TNF-
signaling cascade leading to NF-
B activation. The effects of TNF-
are mediated by its binding to the TNF-R1 and TNF-R2 (69). Binding of
TNF-
to TNF-R1 is responsible for most of the known cellular effects
of TNF-
(79). TRADD is an adaptive protein that binds to TNF-R1.
FADD/MORT-1 binds to TRADD (5, 41) and is responsible for TNF-
induced apoptosis by directly interacting with caspase-8 (FLICE/MACH)
(3). TRADD also interacts with TRAF-2 (41), which is believed to be
responsible for both the activation of JNK (51) through the
intermediary small G proteins Rac and cdc 42 (6) and for the
phosphorylation of I-
B through an I-
B kinase complex (41). TNF-R2
also interacts with TRAF-2 and activates NF-
B (61). TRAF-2 directly
binds to NIK-1 (43), a member of the MAP kinase kinase kinase (MAPKKK) family, which acts as an intermediate molecule in I-
B-
phosphorylation. NIK-1 directly binds I-
B-
kinase (IKK), which in
turn associates with I-
B-
and phosphorylates it on serines 32 and
36 (10, 43). Phosphorylated I-
B-
then undergoes ubiquitination
and degradation, releasing active NF-
B.
In our study, the quiescent HSC responded to incubation with TNF-
with a weak but significant activation of JNK but not of NF-
B. These
results would be most consistent with a functional TNF-R1, TRADD, and
TRAF-2, but with an inhibition of inducible I-
B degradation,
resulting in an intact JNK pathway but not a NF-
B pathway in
quiescent HSC. Although the defective mechanism is unknown, simple
testable hypotheses include that the IKK is not activated because of
the lack of an effective upstream transducer, that constitutive
phosphatase activity dephosphorylates I-
B-
, or that
ubiquitination or proteasome activity is defective. The intactness of
other TNF-
signaling pathways, such as through caspase-8,
phospholipase A2, or phospholipase
C in quiescent vs. activated HSC is unknown.
A switch in the signaling pathway has been reported in pre-B cells
where the major type of NF-B complex is composed of p50/p65 heterodimers, which is in contrast to mature B cells, where p50/cRel is
the major NF-
B complex (48). Although the p65 dimer is the predominant NF-
B protein in activated HSC, our data demonstrate that
signaling pathway switching occurs during HSC transformation, which may
contribute to the expression of a new cellular phenotype and/or
the upregulation of a new set of genes. Acquisition of a functional
I-
B/NF-
B signaling system by activated HSC correlates with the
upregulation of ICAM-1 and MIP-2, suggesting that this process is an
important step in the induction of these newly expressed genes.
Activated HSC express a variety of NF-
B-responsive genes, including ICAM-1 (26), MIP-2 (68), monocyte chemoattractant protein-1
(7, 14, 45, 46), and IL-6 (71).
Increased expression of NF-B-responsive genes such as ICAM-1 and
MIP-2 by activated HSC may have an impact on the development of liver
inflammation. ICAM-1, a member of the immunoglobulin superfamily, is a
cell surface protein that contributes to the cellular adhesion and
transmigration of many leukocytes via interactions with the
2 integrins, LFA-1 (CD11a/CD18) and myelin-associated glycoprotein-1 (D116/CD18), located on the leukocyte cell membrane (9,
44). TNF-
induces ICAM-1 through cooperative interactions of the
transcription factors CCAAT enhancer binding protein (C/EBP) and
NF-
B, whose binding sites are located in the ICAM-1 promoter (29,
60). ICAM-1 expression is increased in vascular and nonvascular cells
during inflammation.
Several liver diseases, including alcoholic hepatitis, are
characterized by neutrophil infiltration, mediated by TNF- (64). The
transmigration of the neutrophils out of the sinusoidal space into the
hepatic parenchyma is mediated by ICAM-1, expressed on the sinusoidal
endothelial cell (72) and activated HSC (26). Our study demonstrates
that activated but not quiescent HSC respond to TNF-
or IL-1
by
increasing ICAM-1 expression. Thus the activated HSC might contribute
to hepatic inflammation through leukocyte transmigration. In that
respect, we have recently demonstrated that blocking NF-
B in
activated HSC prevents the cytokine-induced expression of ICAM-1 and
IL-6 genes (25).
MIP-2, a member of the CXC-chemokine family, is a neutrophil
chemoattractant (22). MIP-2 has recently been demonstrated to be a
major HSC-derived chemokine contributing to neutrophil chemotaxis (68).
TNF- induces MIP-2 in HSC after only 3 days of culture but has a
greater induction after advanced transformation of HSC into
myofibroblast (68). Although differences in the purification and
culturing of HSC may account for the differences in the timing of MIP-2
expression between the two studies, our results provide a mechanism for
the differential effect of TNF-
on quiescent and activated HSC. Thus
TNF-
via NF-
B activation will result in chemoattraction and
transmigration of neutrophils by activated but not by quiescent HSC. An
unknown agonist must provide the initiation of HSC activation to
manifest these effects.
According to this study, the cytokines TNF- and IL-1 produced during
hepatic inflammation should not stimulate NF-
B activity or
NF-
B-dependent gene expression in quiescent HSC. A driving force
behind liver inflammation may be the activation of HSC into NF-
B-responsive cells that then respond to cytokines by producing cell adhesion molecules and chemokines. Thus inhibition of I-
B degradation is a potential target for anti-inflammatory therapy in the
liver and might influence the activation process of HSC following
fibrotic stimuli.
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ACKNOWLEDGEMENTS |
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We thank Dr. Richard Rippe for critical review of this manuscript.
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FOOTNOTES |
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This work was supported by National Institutes of Health Grants GM-41804, DK-47700, and DK-34987, and the Deutsche Forschungsgemeinschaft Grant He-2458/1-1.
C. Hellerbrand and C. Joblin contributed equally to this work.
Address for reprint requests: D. A. Brenner, UNC-CH, Div. of Digestive Diseases and Nutrition, 326 Burnett-Womack Bldg., CB 7080, Chapel Hill, NC 27599-7080.
Received 3 October 1997; accepted in final form 8 April 1998.
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REFERENCES |
---|
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---|
1.
Barnes, P. J.,
and
M. Karin.
Nuclear factor-kappa B: a pivotal transcription factor in chronic inflammatory diseases.
N. Engl. J. Med.
336:
1066-1071,
1997
2.
Beg, A. A.,
T. S. Finco,
P. V. Nantermet,
and
A. S. Baldwin, Jr.
Tumor necrosis factor and interleukin-1 lead to phosphorylation and loss of I kappa B alpha: a mechanism for NF kappa B activation.
Mol. Cell. Biol.
13:
3301-3310,
1993[Abstract].
3.
Boldin, M. P.,
T. M. Goncharov,
Y. V. Goltsev,
and
D. Wallach.
Involvement of MACH, a novel MORT1/FADD-interacting protease, in Fas/APO-1- and TNF receptor-induced cell death.
Cell
85:
803-815,
1996[Medline].
4.
Brenzel, A.,
and
A. M. Gressner.
Characterization of insulin-like growth factor (IGF)-I-receptor binding sites during in vitro transformation of rat hepatic stellate cells to myofibroblasts.
Eur. J. Clin. Chem. Clin. Biochem.
34:
401-409,
1996[Medline].
5.
Chinnaiyan, A. M.,
C. G. Tepper,
M. F. Seldin,
K. O'Rourke,
F. C. Kischkel,
S. Hellbardt,
P. H. Krammer,
M. E. Peter,
and
V. M. Dixit.
FADD/MORT1 is a common mediator of CD95 (Fas/APO-1) and tumor necrosis factor receptor-induced apoptosis.
J. Biol. Chem.
271:
4961-4965,
1996
6.
Coso, O. A.,
M. Chiariello,
J.-C. Yu,
H. Teramoto,
P. Crespo,
N. Xu,
T. Miki,
and
J. S. Gutkind.
The small GTP-binding proteins Rac1 and Cdc42 regulate the activity of the JNK/SAPK signaling pathway.
Cell
81:
1137-1146,
1995[Medline].
7.
Czaja, M.,
A. Geerts,
J. Xu,
P. Schmiedeberg,
and
Y. Ju.
Monocyte chemoattractant protein 1 (MCP-1) expression occurs in toxic rat liver injury and human liver disease.
J. Leukoc. Biol.
55:
120-126,
1994[Abstract].
8.
DeLeeuw, A. M.,
S. P. McCarthy,
A. Geerts,
and
D. L. Knook.
Purified rat liver fat storing cells divide in culture and contain collagen.
Hepatology
4:
392-403,
1984[Medline].
9.
Diamond, M. S.,
D. E. Staunton,
A. R. deFougerolles,
S. A. Stacker,
J. Garcia-Aguilar,
M. L. Hibbs,
and
T. A. Springer.
ICAM-1 (CD 54): a counter-receptor for Mac-1 (CD11b/CD18).
J. Cell Biol.
111:
3129-3139,
1990[Abstract].
10.
Didonato, J. A.,
M. Hayakawa,
D. M. Rothwarf,
E. Zandi,
and
M. Karin.
A cytokine-responsive I(kappa)B kinase that activates the transcription factor NF kappa B.
Nature
388:
548-554,
1997[Medline].
11.
Dignam, J. D.,
R. M. Lebovitz,
and
R. G. Roeder.
Accurate transcription initiation by RNA polymerase II in a soluble extract from isolated mammalian nuclei.
Nucleic Acids Res.
11:
1475-1489,
1983[Abstract].
12.
Essani, N. A.,
G. M. McGuire,
A. M. Manning,
and
H. Jaeschke.
Differential induction of mRNA for ICAM-1 and selectins in hepatocytes, Kupffer cells and endothelial cells during endotoxemia.
Biochem. Biophys. Res. Commun.
211:
74-82,
1995[Medline].
13.
Finco, T. S.,
and
A. S. Baldwin.
Mechanistic aspects of NF kappa B regulation: the emerging role of phosphorylation and proteolysis.
Immunity
3:
263-272,
1995[Medline].
14.
Freter, R. R.,
J. A. Alberta,
G. Y. Hwang,
A. L. Wrentmore,
and
C. D. Stiles.
Platelet-derived growth factor induction of the immediate-early gene MCP-1 is mediated by NF kappa B and a 90-kDa phosphoprotein coactivator.
J. Biol. Chem.
271:
17417-17424,
1996
15.
Friedman, S. L.
Cellular sources of collagen and regulation of collagen production in liver.
Semin. Liver Dis.
10:
20-29,
1990[Medline].
16.
Friedman, S. L.
Seminars in medicine of the Beth Israel Hospital, Boston. The cellular basis of hepatic fibrosis. Mechanisms and treatment strategies.
N. Engl. J. Med.
328:
1828-1835,
1993
17.
Friedman, S. L.,
D. C. Rockey,
R. F. McGuire,
J. J. Maher,
J. K. Boyles,
and
G. Yamasaki.
Isolated hepatic lipocytes and Kupffer cells from normal human liver: morphological and functional characteristics in primary culture.
Hepatology
15:
234-243,
1992[Medline].
18.
Friedman, S. L.,
G. Yamasaki,
and
L. Wong.
Modulation of transforming growth factor beta receptors of rat lipocytes during the hepatic wound healing response.
J. Biol. Chem.
269:
10551-10558,
1994
19.
Furutani, M.,
S. Arii,
K. Monden,
Y. Adachi,
N. Funaki,
H. Higashitsuji,
S. Fujita,
M. Mise,
S. Ishiguro,
and
T. Kitao.
Immunologic activation of hepatic macrophages in septic rats: a possible mechanism of sepsis-associated liver injury.
J. Lab. Clin. Med.
123:
430-436,
1994[Medline].
20.
Gong, W.,
S. Roth,
K. Michel,
and
A. M. Gressner.
Isoforms and splice variant of transforming growth factor beta-binding protein in rat hepatic stellate cells.
Gastroenterology
114:
352-363,
1998[Medline].
21.
Gressner, A. M.
Mediators of hepatic fibrogenesis.
Hepatogastroenterology
43:
92-103,
1996[Medline].
22.
Haelens, A.,
P. Proost,
S. Struyf,
G. Opdenakker,
and
J. van Damme.
Leukocyte migration and activation by murine chemokines.
Immunobiology
195:
499-521,
1996[Medline].
23.
Hart, R. P.,
C. Liu,
A. M. Shadiack,
R. J. McCormack,
and
G. M. Jonakait.
An mRNA homologous to interleukin-1 receptor type I is expressed in cultured rat sympathetic ganglia.
J. Neuroimmunol.
44:
49-56,
1993[Medline].
24.
Haskill, S.,
A. A. Beg,
S. M. Tompkins,
J. S. Morris,
A. D. Yurochko,
A. Sampson-Johannes,
K. Mondal,
P. Ralph,
and
A. S. Baldwin, Jr.
Characterization of an immediate-early gene induced in adherent monocytes that encloses I kappa B-like activity.
Cell
65:
1281-1289,
1991[Medline].
25.
Hellerbrand, C.,
C. Jobin,
Y. Iimuro,
L. L. Licato,
R. B. Sartor,
and
D. A. Brenner.
Inhibition of NF kappa B in activated hepatic stellate cells by proteasome inhibitors and an I kappa B super-repressor.
Hepatology.
27:
1285-1295,
1998[Medline].
26.
Hellerbrand, C.,
S. C. Wang,
H. Tsukamoto,
D. A. Brenner,
and
R. A. Rippe.
Expression of intercellular adhesion molecule 1 by activated hepatic stellate cells.
Hepatology
24:
670-676,
1996[Medline].
27.
Henkel, T.,
T. Machleidt,
I. Alkalay,
M. Kronke,
Y. Ben-Neriah,
and
P. A. Baeuerle.
Rapid proteolysis of I kappa B alpha is necessary for activation of transcription factor NF kappa B.
Nature
365:
182-185,
1993[Medline].
28.
Himmler, A.,
I. Maurer-Fogy,
M. Kronke,
P. Scheurich,
K. Pfizenmaier,
M. Lantz,
I. Olsson,
R. Hauptmann,
C. Stratowa,
and
G. R. Adolf.
Molecular cloning and expression of human and rat tumor necrosis factor receptor chain (p60) and its soluble derivative, tumor necrosis factor-binding protein.
DNA Cell Biol.
9:
705-715,
1990[Medline].
29.
Hou, J.,
V. Baichwal,
and
Z. Cao.
Regulatory elements and transcription factors controlling basal and cytokine-induced expression of the gene encoding intercellular adhesion molecule 1.
Proc. Natl. Acad. Sci. USA
91:
11641-11645,
1994
30.
Housset, C.,
D. C. Rockey,
and
D. M. Bissell.
Endothelin receptors in rat liver: lipocytes as a contractile target for endothelin 1.
Proc. Natl. Acad. Sci. USA
90:
9266-9270,
1993[Abstract].
31.
Jaattela, M.
Overexpression of major heat shock protein hsp70 inhibits tumor necrosis factor-induced activation of phospholipase A21.
J. Immunol.
151:
4286-4294,
1993
32.
Jobin, C.,
and
J. Gauthier.
Differential effects of cell density on 5-lipoxygenase (5-LO). Five-lipoxygenase-activating protein (FLAP) and interleukin-1 beta (IL-1 beta) expression in human neutrophils.
Inflammation
21:
235-250,
1997[Medline].
33.
Jobin, C.,
S. Haskill,
R. J. Mayer,
A. Panja,
and
R. B. Sartor.
Evidence for an altered regulation of I kappa B alpha degradation in human colonic epithelial cells.
J. Immunol.
158:
226-234,
1997[Abstract].
34.
Jobin, C., C. Hellerbrand, L. L. Licato, D. A. Brenner, and R. B. Sartor. NF kappa B mediates
cytokine-induced expression of ICAM-1 in an intestinal epithelial cell
line, a process blocked by proteasome inhibitors.
Gut. In press.
35.
Kita, Y.,
T. Takashi,
T. Tamatani,
M. Miyasaka,
and
T. Horiuchi.
Sequence and expression of rat ICAM-1.
Biochim. Biophys. Acta
1131:
108-110,
1992[Medline].
36.
Kurose, I.,
H. Saito,
S. Miura,
H. Ebinuma,
H. Higuchi,
N. Watanabe,
S. Zeki,
T. Nakamura,
M. Takaishi,
and
H. Ishii.
CD18/ICAM-1 dependent oxidative NF kappa B activation leading to nitric oxide production in rat Kupffer cells co-cultured with syngeneic hepatoma cells.
J. Clin. Invest.
99:
867-878,
1997
37.
Kyriakis, J. M.,
P. Banerjee,
E. Nikolakaki,
T. Dai,
E. A. Rubie,
M. F. Ahmad,
J. Avruch,
and
J. R. Woodgett.
The stress-activated protein kinase subfamily of c-Jun kinases.
Nature
369:
156-160,
1994[Medline].
38.
Larrick, J. W.,
and
S. C. Wright.
Cytotoxic mechanism of tumor necrosis factor alpha.
FASEB J.
4:
3215-3223,
1990[Abstract].
39.
Ledebur, H. C.,
and
T. P. Parks.
Transcriptional regulation of the intercellular adhesion molecule-1 gene by inflammatory cytokines in human endothelial cells.
J. Biol. Chem.
270:
933-943,
1995
40.
Lee, K. S.,
M. Buck,
K. Houglum,
and
M. Chojkier.
Activation of hepatic stellate cells by TGF alpha and collagen type I is mediated by oxidative stress through c-myb expression.
J. Clin. Invest.
96:
2461-2468,
1995[Medline].
41.
Liu, Z. G.,
H. Hsu,
and
D. V. Goeddel.
Dissection of TNF receptor 1 effector function: JNK activation is not linked to apoptosis while NF kappa B activation prevents cell death.
Cell
87:
565-576,
1996[Medline].
42.
Mak, K. M.,
M. A. Leo,
and
C. S. Lieber.
Alcoholic liver injury in baboon: transformation of lipocytes to transitional cells.
Gastroenterology
87:
188-200,
1984[Medline].
43.
Malinin, N. L.,
M. P. Boldin,
A. V. Kovalenko,
and
D. Wallach.
MAP3K-related kinase involved in NFkB induction by TNF, CD95 and IL-1.
Nature
385:
540-544,
1997[Medline].
44.
Marlin, S. D.,
and
T. A. Springer.
Purified intercellular adhesion molecule-1 (ICAM-1) is a ligand for lymphocyte function associated antigen 1 (LFA-1).
Cell
51:
813-819,
1987[Medline].
45.
Marra, F.,
G. Grandaliano,
A. J. Valente,
and
H. E. Abboud.
Thrombin stimulates proliferation of liver fat-storing cells and expression of monocyte chemotactic protein-1: potential role in liver injury.
Hepatology
22:
780-787,
1995[Medline].
46.
Marra, F.,
G. Grandaliano,
A. J. Valente,
M. Pinzani,
and
H. E. Abboud.
Cultured human liver fat-storing cells produce monocyte chemotactic protein-1. Regulation by proinflammatory cytokines.
J. Clin. Invest.
92:
1674-1680,
1993[Medline].
47.
McClain, C.,
D. Hill,
J. Schmidt,
and
A. M. Diehl.
Cytokines and alcoholic liver disease.
Semin. Liver Dis.
13:
170-182,
1993[Medline].
48.
Miyamoto, S.,
M. J. Schmitt,
and
I. M. Verma.
Qualitative changes in the subunit composition of kappa B binding complexes during murine B-cell differentiation.
Proc. Natl. Acad. Sci. USA
91:
5056-5060,
1994[Abstract].
49.
Morita, M.,
Y. Watanabe,
T. Akaike,
and
L. A. Aarden.
Inflammatory cytokines up-regulate intercellular adhesion molecule-1 expression on primary cultured mouse hepatocytes and T-lymphocyte adhesion.
Hepatology
19:
426-431,
1994[Medline].
50.
Nakatsukasa, H.,
P. Nagy,
R. P. Evarts,
H. Chu-Chieh,
E. Marsden,
and
S. S. Thorgeirsson.
Cellular distribution of transforming growth factor beta 1 and procollagen types I, III, and IV transcripts in carbon tetrachloride-induced rat liver fibrosis.
J. Clin. Invest.
85:
1833-1843,
1990[Medline].
51.
Natoli, G.,
A. Costanzo,
A. Ianni,
D. J. Templeton,
J. R. Woodgett,
C. Balsano,
and
M. Levrero.
Activation of SAPK/JNK by TNF receptor 1 through a noncytotoxic TRAF2-dependent pathway.
Science
275:
200-203,
1997
52.
Nudel, U.,
R. Zakut,
M. Shani,
S. Neuman,
Z. Levy,
and
D. Yaffe.
The nucleotide sequence of the rat cytoplasmic beta-actin gene.
Nucleic Acids Res.
11:
1759-1771,
1983[Abstract].
53.
Ogawa, K.,
J.-I. Suzuki,
H. Mukai,
and
M. Mori.
Sequential changes of extracellular matrix and proliferation of Ito cells with enhanced expression of desmin and actin in focal hepatic injury.
Am. J. Pathol.
125:
611-619,
1986[Abstract].
54.
Ohira, H.,
T. Ueno,
S. Shakado,
M. Sakamoto,
T. Torimura,
S. Inuzuka,
M. Sata,
and
K. Tanikawa.
Cultured rat hepatic sinusoidal endothelial cells express intercellular adhesion molecule-1 (ICAM-1) by tumor necrosis factor-alpha or interleukin-1 alpha stimulation.
J. Hepatol.
20:
729-734,
1994[Medline].
55.
Poulos, J. E.,
J. D. Weber,
J. M. Bellezzo,
A. M. DiBisceglie,
R. S. Britton,
B. R. Bacon,
and
J. J. Baldassare.
Fibronectin and cytokines increase JNK, ERK, AP-1 activity, and transin gene expression in rat hepatic stellate cells.
Am. J. Physiol.
273 (Gastrointest. Liver Physiol. 36):
G804-G811,
1997
56.
Ramadori, G.,
T. Weit,
S. Schwogler,
H. P. Dienes,
T. Knittel,
H. Rieder,
and
K.-H. Meyer zum Buchenfelde.
Expression of the gene of the alpha-smooth muscle actin isoform in rat liver and in rat fat-storing (Ito) cells.
Virchows Arch.
59:
349-357,
1990.
57.
Ramm, G. A.,
R. S. Britton,
R. O'Neill,
and
B. R. Bacon.
Identification and characterization of a receptor for tissue ferritin on activated rat lipocytes.
J. Clin. Invest.
94:
9-15,
1994[Medline].
58.
Rippe, R. A.,
G. Almounajed,
and
D. A. Brenner.
Sp1 binding activity increases in activated Ito cells.
Hepatology
22:
241-251,
1995[Medline].
59.
Rockey, D. C.
The cellular pathogenesis of portal hypertension: stellate cell contractility, endothelin, and nitric oxide.
Hepatology
25:
2-5,
1997[Medline].
60.
Roebuck, K. A.,
A. Rahman,
V. Lakshminarayanan,
K. Janakidevi,
and
A. B. Malik.
H2O2 and tumor necrosis factor alpha activate intercellular adhesion molecule 1 (ICAM-1) gene transcription through distinct cis-regulatory elements within the ICAM-1 promoter.
J. Biol. Chem.
270:
18966-18974,
1995
61.
Rothe, M.,
S. C. Wong,
W. J. Henzel,
and
D. V. Goeddel.
A novel family of putative signal transducers associated with the cytoplasmic domain of the 75 kDa tumor necrosis factor receptor.
Cell
78:
681-692,
1994[Medline].
62.
Sakamoto, M.,
T. Ueno,
H. Sugawara,
T. Torimura,
R. Tsuji,
K. Sujaku,
M. Sata,
and
K. Tanikawa.
Relaxing effect of interleukin-1 on rat cultured Ito cells.
Hepatology
25:
1412-1417,
1997[Medline].
63.
Satoh, S.,
A. K. Nussler,
Z. Z. Liu,
and
A. W. Thomson.
Proinflammatory cytokines and endotoxin stimulate ICAM-1 gene expression and secretion by normal human hepatocytes.
Immunology
82:
571-576,
1994[Medline].
64.
Schlayer, H. J.,
H. Laaff,
T. Peters,
M. Woort-Menker,
H. C. Estler,
U. Karck,
H. E. Schaefer,
and
K. Decker.
Involvement of tumor necrosis factor in endotoxin-triggered neutrophil adherence to sinusoidal endothelial cells of mouse liver and its modulation in acute phase.
J. Hepatol.
7:
239-249,
1988[Medline].
65.
Schmitt-Graff, A.,
G. Chakroun,
and
G. Gabbiani.
Modulation of perisinusoidal cell cytoskeletal features during experimental hepatic fibrosis.
Virchows Arch.
422:
99-107,
1993.
66.
Scoazec, J. Y.,
and
G. Feldmann.
In situ immunophenotyping study of endothelial cells of the human hepatic sinusoid: results and functional implications.
Hepatology
14:
789-797,
1991[Medline].
67.
Shiratori, Y.,
Y. Ichida,
A. Geerts,
and
E. Wisse.
Modulation of collagen synthesis by fat-storing cells isolated from CCl4 or vitamin A-treated rats.
Dig. Dis. Sci.
32:
1281-1289,
1987[Medline].
68.
Sprenger, H.,
A. Kaufmann,
H. Garn,
B. Lahme,
D. Gemsa,
and
A. M. Gressner.
Induction of neutrophil-attracting chemokines in transforming rat hepatic stellate cells.
Gastroenterology
113:
277-285,
1997[Medline].
69.
Tartaglia, L. A.,
R. F. Weber,
I. S. Figari,
C. Reynolds,
M. A. Palladino,
and
D. V. Goeddel.
The two different receptors for tumor necrosis factor mediate distinct cellular responses.
Proc. Natl. Acad. Sci. USA
88:
9292-9296,
1991[Abstract].
70.
Thanos, D.,
and
T. Maniatis.
NF kappa B: a lesson in family values.
Cell
80:
529-532,
1995[Medline].
71.
Tiggelman, A. M. B. C.,
W. Boers,
C. Linthorst,
H. S. Brand,
M. Sala,
and
R. A. F. M. Chamuleau.
Interleukin-6 production by human liver (myo)fibroblasts in culture. Evidence for a regulatory role of LPS, IL-1 beta and TNF alpha.
J. Hepatol.
23:
295-306,
1995[Medline].
72.
Volpes, R.,
J. J. van den Oord,
and
V. J. Desmet.
Immunohistochemical study of adhesion molecules in liver inflammation.
Hepatology
12:
59-65,
1990[Medline].
73.
Weiner, F. R.,
M. A. Giambrone,
M. J. Czaja,
A. Shah,
G. Annoni,
S. Takahashi,
M. Eghbali,
and
M. A. Zern.
Ito-cell gene expression and collagen regulation.
Hepatology
11:
111-117,
1990[Medline].
74.
Westwick, J.,
C. Weitzel,
A. Minden,
M. Karin,
and
D. A. Brenner.
Tumor necrosis factor alpha stimulates AP-1 activity through prolonged activation of the c-Jun kinase.
J. Biol. Chem.
269:
26396-26401,
1994
75.
Westwick, J. K.,
and
D. A. Brenner.
Methods for analyzing c-Jun kinase.
Methods Enzymol.
255:
342-359,
1995[Medline].
76.
Widmer, U.,
K. R. Manogue,
A. Cerami,
and
B. Sherry.
Genomic cloning and promoter analysis of macrophage inflammatory protein (MIP)-2, MIP-1 alpha, and MIP-1 beta, members of the chemokine superfamily of proinflammatory cytokines.
J. Immunol.
150:
4996-5012,
1993
77.
Wong, L.,
G. Yamasaki,
R. J. Johnson,
and
S. L. Friedman.
Induction of platelet-derived growth factor receptor in rat hepatic lipocytes during cellular activation in vivo and in culture.
J. Clin. Invest.
94:
1563-1569,
1994[Medline].
78.
Wu, X.,
G. J. Dolecki,
and
J. B. Lefkowith.
GRO chemokines: a transduction, integration, and amplification mechanism in acute renal inflammation.
Am. J. Physiol.
269 (Renal Fluid Electrolyte Physiol. 38):
248-256,
1995.
79.
Yuan, J.
Transducing signals of life and death.
Curr. Opin. Cell Biol.
9:
247-251,
1997[Medline].