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RESEARCH ARTICLE | JUNE 15 1997
Transcriptional activation of vascular cell adhesion molecule-1 gene in vivo
and its role in the pathophysiology of neutrophil-induced liver injury in
murine endotoxin shock.
N A Essani; ... et. al
J Immunol (1997) 158 (12): 5941–5948.
https://doi.org/10.4049/jimmunol.158.12.5941
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Transcriptional Activation
of
Vascular Cell Adhesion
Molecule-1 Gene In Vivo and Its Role in the Pathophysiology
of
Neutrophil-Induced Liver Injury in Murine Endotoxin
Shock'
Naeem A. Essani,* Mary Lynn Bajt,t Anwar Farhood,§ Steven
L.
Vonderfecht,* and
Hartmut Jaeschke2*
Polymorphonuclear leukocytes (neutrophils) can cause hepatic parenchymal cell injury during endotoxin (ET) shock. Because
adhesion molecules are critical for inflammatory cell damage, the role
of
vascular cell adhesion molecule-1 (VCAM-1) was
studied in the pathophysiology of
ET
shock. El-sensitive mice (C3Heb/FeJ) were treated with 700 mg/kg galactosamine in
combination with 100 pg/kg
Salmonella aborfus egui
ET, 15
&kg
TNF-a, or 13 to 23 pg/kg IL-1. VCAM-1 mRNA formation
was strongly activated in animals treated with
ET,
TNF-a, or IL-1. In contrast, only TNF-a and IL-1, not
ET,
induced VCAM-1
gene transcription in livers of ET-resistant mice (C3H/Hej). Immunohistochemistry and isolation of liver cells during endotox-
emia indicated that VCAM-1 mRNA and protein were only formed in endothelial cells and Kupffer cells, not in hepatocytes.
Calactosamine/ET induced neutrophil accumulation in sinusoids (515
&
30 neutrophils/50 high power fields) followed by
transmigration at
7
h.
At that time, severe liver injury was observed (necrosis, 53
2
5%). An anti-VCAM-1 Ab (3 mg/kg)
attenuated the area
of
necrosis by
60%.
The Ab reduced neutrophil transmigration by 84%, but had no effect on the total
number
of
cells in the liver vasculature. Flow cytometric analysis identified the presence of very late Ag-4 on mouse peripheral
neutrophils. Our data demonstrated cytokine-dependent VCAM-1 gene transcription and protein expression in the liver during
endotoxemia. Neutrophils were able to use very late Ag-4/VCAM-1 interactions to transmigrate into liver parenchyma in vivo.
Preventing transmigration by blocking VCAM-1 protected hepatocytes against neutrophil-induced injury.
The
Journal
of
Immunology,
1997, 158: 5941-5948.
V
ascular cell adhesion molecule-l (VCAM-I)Z is a mem-
ber
of
the Ig gene superfamily and can be expressed on
vascular endothelial cells
(I).
The very late Ag-4
(VLA-4;
a4//3,)
is recognized as the counter-receptor of VCAM-
1
(2). VLA-4 is expressed on monocytes (3), lymphocytes (4), eo-
sinophils, and basophils
(5).
Consequently, the adhesion of these
leukocytes
to
vascular endothelium and transmigration are in part
dependent
on
VLA-4/VCAM-I interactions in vitro (3, 6-1 1) and
in vivo (12-15). Until recently, polymorphonuclear leukocytes
(neutrophils; PMNs) have been generally considered to lack
VLA-4 expression
(10).
However, several recent reports provided
'Cardiovascular Pharmacology, +Cell Biology and Inflammation Research, and
*Preclinical Tox~cology, Pharmacia
&
Upjohn, Inc., Kalamazoo, MI 49007; and
4Department of Pathology, University
of
Texas Health Science Center, Houston,
TX 77030
Received for publication December 9, 1996. Accepted
for
publication March
7, 1997.
The
costs
of
publication
of
this article were defrayed in part
by
the payment of
page charges. This article must therefore he hereby marked advertisement in
accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
R01
ES06091.
'
This work was supported in part by National Institute
of
Health Grant
'
Address correspondence and reprint requests to Dr. Hartmut Jaeschke, Cardio-
vascular Pharmacology (7243-300-210), Pharmacia
&
Upjohn, Inc., 301 Hen-
pnu.com
rietta St., Kalamaroo,
MI
49007. E-mail address: 1lartmut.w.jaeschkeQam.
'
Abbreviations used in this paper: VCAM-1, vascular cell adhesion molecule-1;
VLA-4, very late antigen-4; PMN, polymorphonuclear leukocytes; ICAM-1, in-
tercellular adhesion molecule-1;
ET,
endotoxin; Gal, galactosamine; ALT, ala-
nine aminotransferase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase;
HPF, high power field;
NF-KB,
nuclear factor
KB.
Copyrlght
0
19Y7
by
The American Association
of
Imnlunologists
evidence for the presence
of
VLA-4 on human (16) and rat neu-
trophils
(17).
Moreover, VLA-4 can mediate neutrophil infiltration
to inflammatory sites in the skin and joints
in
vivo
(17).
Neutrophil-induced liver injury has been documented during
ischemia reperfusion
(18)
and endotoxemia
(19).
In each of these
disease models, there is up-regulation
of
the
&
integrin Mac-I
(CDI lb/CD18) on neutrophils (20-22) and transcriptional activa-
tion of intercellular adhesion molecule-I (ICAM-I) on sinusoidal
lining cells and hepatocytes (21, 23-26). Moreover, Abs against
CDI
1
b, CD18, and ICAM-I attenuated liver injury in these mod-
els (18-21, 23, 27). However, the Abs had either no etfect or only
a limited impact on neutrophil sequestration
in
the liver vascula-
ture (28). These data led to the hypothesis that the mechanism of
neutrophil-induced liver injury consists of three steps, which in-
clude the sequestration of neutrophils in sinusoids followed by
their transendothelial migration and adherence to parenchymal
cells (29). Whereas the initial neutrophil sequestration
in
the liver
does not require
p2
integrin/ICAM-
1
interactions (28), transmigra-
tion (21) as well as adherence-dependent cytotoxicity (30) are
strongly dependent on
p2
integrins and ICAM-1. In light
of
the
recent findings that neutrophils are also able to use VLA4/
VCAM-
1
for transmigration in the skin and joints
(1
7), we eval-
uated the regulation of VCAM-I expression and its pathophysio-
logic role in a murine endotoxemia model
of
neutrophil-induced
liver injury. Our data document a rapid, cytokine-dependent tran-
scriptional activation of the hepatic VCAM-
1
gene in vivo. Block-
ing VCAM-I with an Ab had no effect on neutrophil sequestration,
but etfectively attenuated transmigration and liver injury. Because
VCAM-
1
expression during endotoxemia is restricted to sinusoidal
0022-1 767/Y7/$02.00
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5942
VCAM-1 GENE ACTIVATION AND ET-INDUCED LIVER
INJURY
lining
cells, we concluded that neutrophils
can
use
VLA-W
VCAM-I
interactions in addition to
p2
integriniICAM-l for ex-
travasation in the liver vasculature. Preventing neutrophil transmi-
gration protects hepatocytes against inflammatory cell
injury.
Materials
and
Methods
Animals
Male mice, strains C3HeblFeJ (endotoxin (ET)-sensitive) and C3WHeJ
(ET-resistant; 20-25
g
body weight), were purchased from The Jackson
Laboratory (Bar Harbor, ME). The animals had free access to food (cer-
tified rodent diet 5002C, PMI Feeds, Inc., Richmond, IN) and water. The
experimental protocols followed the criteria of Pharmacia
&
Upjohn, Inc.,
and the National Research Council for the care and use of laboratory
an-
imals in research. Animals were treated i.p. with 700 mgkg o-galac-
tosamine (o-GaI; Sigma .Chemical Co., St. Louis, MO) and
100
pgkg
Salnzonellu
abortus
equi
ET (Sigma Chemical Co.) dissolved in sterile PBS
VCAM-I mAb (clone 429; PharMingen, San Diego, CA) or the isotype-
(pH 7.0). Some animals were treated with 3 mgkg of the anti-murine
matched rat
IgG
(PharMingen) at the time of GalET injection. All Ah
solutions were low in ET
(50.01
ng/pg protein).
In
some experiments, ET
was replaced by i.v. injection of murine rTNF-a
(15
pgkg;
sp. act., 4
X
10‘
U/mg; Genzyme, Cambridge, MA), murine rIL-la (13 pgikg; sp. act.,
8
X
IO6
Ulmg; Genzyme),
or
murine rIL-Ip (23 pg/kg; sp. act.,
1.5
X
IO6
U/mg; Genzyme).
Experimental protocols
The animals were killed by cervical dislocation at various times after the
administration
of
GalET
or
a cytokine (TNF-a, IL-la,
or
IL-lp). Blood
was collected from the right ventricle into
a
heparinized syringe and cen-
trifuged, and plasma was used for determination of alanine aminotransfer-
ase (ALT) activity with Sigma Chemical Co. test kit DG 159-UV. Pieces
of the liver were immediately homogenized for RNA isolation
as
described
below; other parts of each liver were fixed in phosphate-buffered formalin
for histologic analysis
or
embedded in OCT embedding medium (Miles
Diagnostic Division, Elkhart, IN) and snap-frozen in methylbutane cooled
in liquid nitrogen for immunohistochemistry.
Northern blot analysis
Total cellular RNA was isolated from liver tissue according to a method
described by Chomczynski and Sacchi (31). Freshly excised liver sections
were homogenized in RNAzol (Tel-Test, Inc., Friendswood, TX) and ex-
tracted with guanidine thiocyanate-phenol-chloroform. RNA was collected
by overnight precipitation at 4°C with isopropanol. RNA pellets were
washed twice with ice-cold 75% ethanol (v/v), vacuum-dried, and then
dissolved in
a
small volume of
I
mM EDTA (pH 7.0). Isolated RNA was
stored at -70°C. RNA was quantified spectrophotometrically, and equal
amounts of RNA samples were electrophoresed
on
denaturing agarose-
formaldehyde gels and transferred to Genescreen Plus hybridization mem-
branes (New England Nuclear Research Products, Boston. MA) using the
capillary elution technique
(32).
RNA was cross-linked by baking the
membranes at
80°C
for 2 h under vacuum. A mouse VCAM-I hybridiza-
tion probe was prepared by PCR using
a
cDNA probe prepared for murine
VCAM-I by reverse transcription-PCR using total mouse
lung
RNA
as
a
template (courtesy of Dr. A. M. Manning). The following primer pair
was used: 5’-CCACAAACCAAGCCATGCAT/CGTACCGTACAGTAC
CATGT-3’. The resulting PCR fragments were subcloned in the pBlue-
script vector for propagation and sequence determination. A cDNA probe
was then generated by PCR from plasmid constructs containing the con-
firmed murine sequences
(33).
Mouse glyceraldehyde-3-phosphate dehy-
drogenase (GAPDH) probe was prepared using
a
PCR-Amplimer kit
(Clontech. Palo Alto, CA). Purified fragments were radiolabeled with
[a-”P]dCTP, using
a
random hexanucleotide primer kit (Stratagene, La
Jolla. CA). Transfered membranes were prehybridized with RAPID-Hyb
buffer (Amersham, Arlington Heights,
1L)
at 65°C for 2 h and then hy-
bridized with labeled probes overnight at 65°C. Membranes were washed
in
1
X SSC
(0.15
M
sodium chloride and
0.015
M sodium citrate, pH 7.0)
containing
0.1%
SDS for
15
min at room temperature. Membranes were
washed twice at 55°C in 0.2X SSC containing
0.1%
SDS for
30
rnin.
The washed blots were exposed to Hyperfilm MP x-ray film (Amer-
sham) at -80°C.
Flow cytometric analysis
Peripheral blood neutrophils were stained using Coulter Immunology’s
(Hialeah, FL) whole blood lysis kit according to the manufacturer‘s in-
structions, Briefly.
100
FI
of whole blood was washed three times with
PBS containing
0.1%
BSA (PBS/BSA). Cells were resuspended with
100
pl
of PBS/BSA containing
1
pg
of
FITC-conjugated RB6-8CS (anti-
GR-1) and phycoerythrin-conjugated M1/70 (anti-a,)
or
phycoerythrin-
conjugated RI-2 (anti-a,) Abs (PharMingen). Following incubation for 30
min on ice, cells were pelleted by centrifugation and washed twice with
PBS. Cells were lysed with
1
ml of Immuno-Lyse (Coulter Corp., Miami,
FL) for 2 min at room temperature and fixed with 250
p1
of Coulter clone
fixative. Two-color analysis of Ab binding to cells was performed using a
Coulter EPICS Elite ESP flow cytometer. Peripheral blood neutrophils
were gated by the forward
and
light angle scatter and GR-I FITC fluores-
cence. Nonspecific fluorescence was determined on cells incubated with
isotype- and cytochrome-matched control Abs.
Histology
Formalin-fixed portions of the liver were paraffin embedded, and 5-~m-
thick sections were cut. PMNs were stained employing the AS-D chloro-
acetate esterase technique as described in detail previously (28). PMNs
were identified by positive staining and morphology and were counted in
50
high power fields (HPF; X400) using
a
Nikon Labophot microscope
(Nikon Corp., Melville, NY). Only those neutrophils present within sinu-
soids
or
extravasated into the tissue were counted. Neutrophils present in
large hepatic vessels, e.g., venules, were not counted. The percentage
of
necrotic area was estimated by evaluating parallel sections stained with
hematoxylin and eosin. The pathologist (A.F.) performing the histologic
evaluation (PMN, area of necrosis) was blinded
as
to the treatment of the
animals.
immunohistochemistry
Frozen liver specimens were sectioned at
6
pm in
a
cryostat; sections were
placed
on
ProbeOn glass microscope slides (Fisher Scientific, Pittsburgh,
PA), air-dryed for
15
min, fixed in acetone for
1
min, and air-dried for
15
min. Air-drying and acetone fixation were performed at room temperature.
Slides were stored at -70°C in an air-tight container until needed for
immunohistochemical staining. All subsequent steps were performed
at
room temperature using the Microprobe slide handling system (Fisher).
Wash buffer consisted
of
PBS (Dulbecco’s; pH 8) containing 0.25% Brij
35
solution (Sigma Chemical Co., St. Louis, MO). Sections were first brought
to room temperature, then incubated for
IO
min with 0.28 neutral buffered
formalin in wash buffer, and briefly rinsed three times with wash buffer.
They were then covered with wash buffer containing
IO%
fetal bovine
serum for
10
min, followed by 30 min with either rat mAb to mouse
VCAM-
1
(clone 429 MVCAM.A, PharMingen)
or
rat
IgC,,
(PharMingen)
diluted in wash buffer containing
10%
fetal bovine serum. The slides were
rinsed quickly three times with wash buffer, then three times with absolute
methanol, followed by 5-min incubation in absolute methanol containing
1%
H202.
Sections were again rinsed quickly three times with absolute
methanol, followed by three rinses with wash buffer, and 30-mi, incuba-
tion in peroxidase-labeled F(ab’), fragments
of
mouse mAb to rat
IgG
(Jackson ImmunoResearch Laboratories, West Grove, PA). The peroxi-
dase-laheled Ab was diluted in wash buffer containing
10%
fetal bovine
serum. Slides were rinsed quickly three times in wash buffer, and the
la-
beled Ab remaining bound to the sections was detected using the chroma-
gen, diaminobenzidine (Sigma Chemical Co.). Sections were counter-
stained with methyl green/Alcian blue (Sigma Chemical Co.), covered with
a
coverslip, and examined by light microscopy. The intensity of staining
was scored by the examiner without knowledge of the treatment groups
according to the amount of brown stain precipitate
(0
=
none.
I
=
mild.
2
=
moderate, 3
=
prominent).
isolation
of
rat liver cells
Cells were isolated from male Fischer rats (240-290 g body weight; Harlan
Sprague-Dawley. Inc.. Indianapolis, IN). The animals were treated with an
i.v. injection of
0.5
mg/kg
Salmonella
enferifidis
ET (Sigma Chemical Co.,
St. Louis, MO)
or
I
ml/kg saline under light ether anesthesia. After
1
h the
animals were anesthetized with pentobarbital. Hepatocytes were isolated
after collagenase digestion, and hepatic endothelial cells and Kupffer cells
were separated by centrifugal elutriation as described in detail previously
(20, 24, 27). Each cell fraction was washed repeatedly with HBSS and was
295% pure
as
assessed by morphology, peroxidase staining, and superox-
ide formation. Cell viability for each cell fraction was
>90%
for hepato-
cytes and >95% for Kupffer cells and endothelial cells
as
determined by
trypan blue exclusion. Immediately after isolation, RNA
was
isolated from
the individual cell fractions
as
described above for mouse liver.
A
rat
VCAM-I cDNA fragment was isolated by PCR from first-strand cDNA
synthesized from total RNA isolated from rat heart tissue after ET treat-
ment (34). Gene-specific oligonucleotides were designed based on the
Downloaded from http://journals.aai.org/jimmunol/article-pdf/158/12/5941/1075894/5941.pdf by guest on 23 December 2022
The
Journal
of
Immunology
1.5
h 4h 7h
FIGURE
1.
Analysis
of
mRNA levels for
VCAM-1 and GAPDH in livers of C3Heb/FeJ
(ET-sensitive) mice
(top graph)
and C3H/HeJ
(ET-resistant) mice
(bottom graph).
Levels of
mRNA were assessed by Northern hybridiza-
tion
in
livers from untreated control animals
(CTL) and at various time points after i.p. ad-
ministration of
700
mgkg Gal
(G),
100
pgkg
S.
abortus
equi
ET
(E),
or a combination
of
Gal
and ET (G&E). Three representative animals
are shown for each group and time point.
CTL'
G
E G&E' G
E
G&E'
G
E
G&E'
I I
1
I
1
I
I I
I
1
VCAM-1
"0"-
"
"-
-00
ET-Sen
1.5
h 4h 7h
I
I I
1
CTL G E G&E G E G&E G G&E
I
I
I
I
I
I
I
I
I
I
1
VCAM-1
5943
3.2
kB
1.4
kB
3.2
kB
GAPDH
1.4
kB
ET-Res
available cDNA sequence for rat VCAM-I (33).
The
resulting PCR prod-
uct was subcloned, and
its
authenticity was confirmed by DNA sequence
analysis. For use in hybridization protocols, DNA fragments were prepared
from this cDNA clone by the
PCR.
Briefly, gene-specific oligonucleotides
were designed to generate fragments of approximately
500
bp from the
VCAM-I cDNA clone. The following oligonucleotide pair was used
(5'oligo/3'oligo; each sequence as
5'
to
3'):
CCAAGCTATGCATTCA
GACTKTGAAAGTCAACCCAGTGAC
encompassing the 3' untrans-
lated region
(34).
The probe for the metabolic enzyme glyceraldehyde-3-
phosphate dehydrogenase (GAPDH) was
a
1.2-kb
fsfl
insert fragment
from
a
plasmid containing the rat GAPDH cDNA. Purified fragments were
radiolabeled with [a-"P]dCTP using
a
random hexanucleotide primer kit
(Stratagene,
La
Jolla, CA) to
a
sp. act. of
IO''
dpdpg.
Statistics
All data are given
as
the mean
2
SE.
Statistical significance between the
control group and
a
treated group was determined with
the
unpaired Stu-
dent's
f
test
or Wilcoxon rank sum test. Comparisons between multiple
groups were performed using one-way analysis of variance followed by
Bonferroni's
f
test.
p
<
0.05 was considered significant.
Results
Northern blot analysis of control livers showed only minor
VCAM-I mRNA expression
in
ET-sensitive mice (Fig.
I).
Ad-
ministration of ET with
or
without Gal rapidly induced VCAM-I
gene transcription. However, mRNA levels were maintained
longer
in
the GallET group than
in
the group with ET alone (Fig.
1).
Gal alone had only a minor effect on hepatic VCAM-I mRNA
levels at later time points. In contrast, the ET-resistant strain did
not respond to ET, GalET,
or
Gal administration with VCAM-I
mRNA formation (Fig.
I).
The major difference between the two
strains is that macrophages of ET-resistant mice do not generate
cytokines (TNF-a and
IL-I)
upon exposure to ET
(35,
36).
Thus,
the lack of VCAM-1
mRNA
expression
in
the ET-resistant strain
suggests that cytokines may be responsible for transcriptional ac-
tivation of the VCAM-I gene
in
the liver. To directly test this
hypothesis, TNF-a
or
IL-l was injected
i.v.
in
Gal-treated animals
of the ET-sensitive and ET-resistant strain (Figs.
2
and
3).
Admin-
istration of TNF-a induced a rapid expression of VCAM-I mRNA
in
both strains; the increased mRNA levels were sustained up to
7
h.
with
slightly higher levels
in
the sensitive strain (Fig.
2).
IL-la
also induced substantial hepatic VCAM-I mRNA formation at
1.5
h,
which was still above baseline levels at 7
h
in
both strains
(Fig.
3).
Similar results were found
with
IL-IP
(data not shown).
G/TNF-a G/TNF-a
1.5
h
7
h
1.5
h
7
h
-
-
-
-
VCAM-1
"
"-0
GAPDH
ET-Sen ET-Res
FIGURE
2.
Analysis
of
mRNA levels for VCAM-1 and GAPDH
in
livers of C3Heb/FeJ (ET-sensitive) mice and C3H/HeJ (ET-resistant)
mice. Levels
of
mRNA were assessed by Northern hybridization 1.5
and
7
h
after the combined administration
of
700
mgAg Gal (i.p.)
and
15
pgkg
of murine rTNF-o! (i.v.). Three representative animals are
shown for each group and time point.
To support the hypothesis that the transcriptional activation of
the VCAM-I gene results
in
increased protein expression, frozen
sections of livers were stained with a mAb to mouse VCAM-
1.
In
livers of untreated animals, only a very weak staining of sinusoidal
lining cells was observed. Larger blood vessels and bile duct ep-
ithelium stained moderately for VCAM-I expression (Fig.
4A).
In
contrast,
4
h
after ET administration there was a substantial
in-
crease
in
VCAM-I expression on sinusoidal lining cells and a
notable, but less prominent, increase in the endothelium of blood
vessels (Fig.
4B).
Samples from a time-course experiment were
then stained, and staining intensity was graded without knowledge
of the treatment groups on a scale of
0
to
3
(0
=
no staining,
1
=
mild,
2
=
moderate,
3
=
prominent). Since changes in the staining
of sinusoidal lining cells were clearly the most prominent feature
of the immunohistochemical evaluations, the staining score reflects
primarily the intensity of staining
in
this compartment. GalET
or
ET alone significantly induced VCAM-
1
protein expression
in
the
liver (Fig.
5).
The increase was only observed
4
and 7
h
after ET.
There was no difference between ET and GallET
in
the onset of
VCAM-
1
protein expression
or
the maximal levels reached at 7
h.
However, the initial increase
in
VCAM-I expression appeared to
be higher
in
GallET livers (Fig.
5).
Gal treatment alone did not
induce VCAM-I expression on any liver cell type within the 7-h
observation period (data not shown).
Although immunohistochemistry clearly demonstrated the ex-
pression of VCAM-I on sinusoidal lining cells,
it
did not allow
us
to completely rule out some minor expression on hepatocytes. To
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5944
VCAM-1 GENE ACTIVATION AND ET-INDUCED LIVER
INJURY
FIGURE
3.
Analysis of mRNA levels for VCAM-1
and CAPDH in livers of C3Heb/FeJ (ET-sensitive)
mice and C3H/HeJ (ET-resistant) mice. Levels
of
mRNA were assessed by Northern hybridization
VCAM-1
1.5 and
7
h after administration of
700
rnglkg Gal
(i.p.) alone or in combination with 13 pglkg
of
mu-
rine rk-1
a.
Three representative animals are shown
GAPDH
for each group and time point.
1.5
h
7h
I
I
1
I
I I I
G
GAL-la
G
WIL-la
FIGURE
4.
Frozen sections
of
livers were stained with a rat mAb to
mouse VCAM-1 (clone 429). The section
of
a control liver
(A)
shows
moderate staining
of
epithelium of a bile duct (large arrow) and en-
dothelium
of
a small artery (small arrow) and vein (v). There
is
only
weak staining of sinusoidal lining cells. This
is
best observed in the
lower right
quadranr
of
the photograph.
B,
Liver section
of
a C3Heb/
FeJ
mouse 4
h
after injection of 100 pgkg
S.
abortus
equi ET, There
is
moderate staining
of
the bile duct (arrow) and prominent staining
of
a
small vein (v). The remainder of the photograph shows the extensive
staining
of
the sinusoidal lining cells for VCAM-1. Bar
=
60
pm.
clarify
this
issue, endothelial cells, Kupffer cells, and hepatocytes
were isolated from controls and from treated cells
1
h
after ad-
ministration of
ET.
Because the isolation procedure was estab-
lished for rat tissue, these experiments were performed
in
rats.
Previous experiments showed that rat and mouse livers respond
similarly
to
administration of
ET
with
regard
to
cytokine formation
and up-regulation of the transcription factor NF-KB and adhesion
molecules such as ICA"1 and selectins
(21,
24,
37,
38).
There
was no basal expression of VCA"1 mRNA in liver cells iso-
lated from control livers (Fig.
6).
ET
treatment substantially
induced VCAM-1 mRNA formation in endothelial cells and
1.5
h
7h
I
I
I
G
GAL-la
G
WIL-la
I
I
1
I
i
ET-Sen
Staining
Score
3
ET-Res
*
T
Control
1.5h
4h
7h
FIGURE
5.
Frozen liver sections of a time-course experiment were
stained with a rat mAb to mouse VCAM-1 (clone 429) as described in
Materials and Methods, and the staining intensity was graded without
knowledge of the treatment groups on a scale of
0
to
3
(0
=
no stain-
ing,
1
=
mild, 2
=
moderate,
3
=
prominent). Mice from the C3Hebl
FeJ strain received
100
&kg
S.
abortus equi ET (ET) either alone or in
combination with
700
mgkg Gal (Gal/ET). Data represent the mean
?
SE
of
five animals per group and time point.
*
indicates
p
<
0.05
(compared with control);
#
indicates
p
<
0.05 (ET vs Cal/ET).
Downloaded from http://journals.aai.org/jimmunol/article-pdf/158/12/5941/1075894/5941.pdf by guest on 23 December 2022
The
Journal
of
Immunology
Hepatocytes
Endothelial
Cells
Kupffew
Cells
5945
C
I
hr
Et
C
1
hr
Et
C
I
hr
Et
"
"
"
VCAM-1
-
00
3.2
kB
OAPDH
-
oo.)".)-
~0000~0~
1AkB
FIGURE
6.
Analysis of mRNA levels for VCAM-1 and GAPDH in hepatic endothelial cells, Kupffer cells, and hepatocytes. Cells were isolated
from livers of male Fisher rats treated with
1
ml/kg saline (C) or
0.5
mgkg
S.
enteritidis ET
(Et)
for
1
h. Results from four animals are shown for
each group.
6000
4500
3000
1500
0
60
45
30
15
0
ALT
U/I
750
500
250
0
G/ET
"
c
G/ET
G/ET
'gG CL429
Necrosis
%
G/ET
C
G/ET
G/ET
IgG CL429
FIGURE
7.
Liver injury,
as
assessed by plasma ALT activities
(A)
and
histologically by the area of necrosis
(6).
was evaluated in C3Heb/FeJ
mice
7
h after the combined administration of
700
mgkg Gal
(G)
and
100
pgkg
S.
abortus equi
ET
(ET). Animals were treated with 3 mgkg
murine VCAM-1 Ab (clone
429)
or isotype-matched control
IgG
at
the
time of G/ET injection. Data represent the mean
?
SE
of
10
animals/
group.
*
indicates
p
<
0.05
(compared with control or
IgG).
was detected, showed that the total number
of
neutrophils (520
2
42 PMN/SO HPF;
n
=
5)
was also unaffected by the anti-VCAM-
I
Ab
or
control IgG.
Because our data suggest that VCAM-I may be involved in
neutrophil transmigration, mouse neutrophils were evaluated by
flow cytometry for expression
of
its known counter-receptor,
VLA-4
(a4//3,).
As demonstrated in Figure 9,
a4
was present on
circulating mouse neutrophils. Approximately 80% of the GR-I
(anti-granulocyte marker)-positive peripheral blood neutrophils
expressed
a4
(CD49d). These neutrophils were also
100%
positive
for
aM
(CDI Ib) expression (Fig. 9). Thus, mouse neutrophils have
PMN
Transmigration
50HPF
T
IT
%
45
30
15
0
G/ET
G/ET G/ET
G/ET
G/ET
G/ET
C
IgG
CL429
C
IgG
CL429
FIGURE
8.
Hepatic neutrophil sequestration and transmigration
were evaluated in C3Heb/FeJ mice
7
h after the combined adminis-
tration of 700 mgkg Gal
(G)
and 100
pgkg
S.
abortus equi ET
(ET).
Animals were treated with 3 mgkg murine VCAM-1 Ab (clone
429)
or
isotype-matched control
IgG
at
the time of G/ET injection. Neutrophils
were counted in
50
HPF. Transmigrated neutrophils are given as
a
percentage of the total neutrophils in the liver. Data represent mean
5
SE
of 10 animals/group.
Fluorescence Intensity
FIGURE
9.
Flow cytometric analysis of mouse neutrophils. Mouse
whole blood was stained with the anti-granulocyte marker GR-1 and
anti-a, (left, broken line) or anti-a, (right, broken line) Abs and ana-
lyzed by flow cytometry. Peripheral neutrophils were gated as de-
scribed in Materials and Methods. The solid line represents gated neu-
trophils stained with isotype-matched control Abs. Results are
depicted
as
histograms, with the
log
of the fluorescence intensity on
the abscissa and the cell number on the ordinate.
the necessary adhesion receptor to recognize and interact with
VCAM-I on sinusoidal lining cells.
Discussion
The principal objective of this investigation was to study the tran-
scriptional regulation
of
the VCAM-I gene in the liver in vivo and
to investigate a potential pathophysiologic role
of
VCAM-I in an
experimental model
of
neutrophil-induced liver injury. Our data
show that there
is
only minimal VCAM-I mRNA and protein
ex-
pression in control livers. However, administration
of
ET drasti-
cally induced mRNA formation and VCAM-I expression in livers
Downloaded from http://journals.aai.org/jimmunol/article-pdf/158/12/5941/1075894/5941.pdf by guest on 23 December 2022
5946
VCAM-1 GENE ACTIVATION AND ET-INDUCED LIVER
INJURY
Of ET-sensitive animals. Immunohistochemistry and mRNA eval-
uation in isolated liver cells after in vivo ET treatment indicate that
VCAM-1 gene activation occurs only in vascular endothelial cells
and Kupffer cells. These findings are similar to those reported for
human livers. VCAM-1 protein expression on sinusoidal lining
cells and Kupffer cells was described in livers of patients during
allograft rejection (391, viral hepatitis (40), and alcoholic cirrhosis
(41). Consistent with
our
in vivo findings in a murine model,
VCAM-1 was not detected on human hepatocytes (39-41). In
contrast to these data, a recent paper reported that prolonged cy-
tokine treatment of murine hepatocytes in vitro induced VCAM-1
mRNA expression (42). However, compared with mRNA levels of
control genes such as p-actin, VCAM-1 expression was extremely
low. Thus, if VCAM-1 gene activation occurs at all in parenchy-
mal cells in the liver in vivo, it appears to be negligible compared
with the expression of other adhesion molecules, e.g., ICAM- 1, on
hepatocytes (23, 24, 26).
VCAM-1 gene expression was only induced in livers of ET-
sensitive animals and was completely absent in ET-resistant ani-
mals. The major difference between these strains is that ET-resis-
tant animals do not generate cytokines in response to ET (35, 36).
These data suggest that the transcriptional activation of the
VCAM-1 gene in the liver in vivo is strictly cytokine dependent.
Administration of TNF-a, IL-1 a, and IL-lp rapidly induced
VCAM-1 mRNA formation in ET-resistant as well as ET-sensitive
cells. This demonstrated that each of these cytokines is able to
activate transcription of the VCAM- 1 gene in livers of animals of
both strains. Because substantial amounts of TNF-a and IL-1 are
generated in this model (Zl), these cytokines may be the major
mediators of the ET effect. Therefore,
our
in vivo data are consis-
tent with previous in vitro findings that a variety of cytokines are
able to induce the VCAM-1 gene in isolated endothelial cells of
variable sources (1, 2, 5, 6,
43).
Furthermore, VCAM-1 gene ac-
tivation is dependent on the transcription factor NF-KB (44, 45).
NF-KB activation was shown in the liver in vivo after ET admin-
istration in rats (37) and mice (38). The cell types involved in
ET-induced NF-KB activation in vivo include hepatic endothelial
cells and Kupffer cells (37).
After documenting the extensive cytokine-dependent transcrip-
tional activation of the VCAM-1 gene in sinusoidal lining cells of
the liver, the main question remained of whether the expression of
this adhesion molecule plays a relevant role in the pathophysiology
of a neutrophil-induced injury mechanism. Similar to previously
reported data for human and rat neutrophils (16, 17), we could
demonstrate the constitutive expression of VLA-4, the known
counter-receptor for VCAM-1, on circulating mouse neutrophils.
This suggests that neutrophils should principally be able
to
interact
with endothelium expressing VCAM-1. Consequently, a mAb to
mouse VCAM-1 significantly attenuated liver injury in GalET-
treated animals. The liver injury in this model is dependent on
neutrophils, as demonstrated by the beneficial effects of Abs block-
ing
p2
integrins (19) and ICAM-1 (21). The initial step in the
mechanism of neutrophil-induced parenchymal cell injury is the
sequestration
of
these leukocytes in sinusoids; this process is in-
dependent of
p2
integrinllCAM-1 interactions (28). The subse-
quent transmigration step and the adherence to parenchymal cells
can be attenuated by blocking
p2
integrins and ICAM-1 in vivo
(21) and in vitro (30). The Ab to VCAM-1 did not affect the total
number of neutrophils in the liver before or after the development
of liver injury. This indicates that neutrophil sequestration in he-
patic sinusoids does not require VLA-4NCAM-1 interactions and
suggests that the beneficial effect of the Ab is not due to preventing
hepatic neutrophil accumulation. However, the anti-VCAM-1 Ab
strongly attenuated transmigration, thereby preventing to a large
degree the attack on parenchymal cells. Because VCAM-1 was not
detected on hepatocytes, our data suggest that the reduced trans-
migration of neutrophils is responsible for the protective effect
of
the anti-VCAM-1 Ab. Thus, neutrophils appear to be able
to
use
VLA-4NCAM-1 in addition to
p2
integrinsllCAM-1 for transmi-
gration in the liver. These observations are similar to those re-
ported by Issekutz et al. in rat models of dermal inflammation and
arthritis (17). In both of these models, VLA, can mediate neutro-
phil transendothelial migration and appears to be able to substitute
for LFA-1 (17). These findings add to a growing number of reports
demonstrating that neutrophils (17,46-48), similar to monocytes
(7,
12, 14, 15, 48), can use multiple adhesion receptors for trans-
migration. An interesting aspect of liver inflammation is the fact
that only neutrophils sequestered in sinusoids, and not those mar-
ginated in postsinusoidal venules, actually transmigrate (49).
Therefore, the expression of VCAM- 1 on sinusoidal lining cells is
more important
for
the pathophysiology of neutrophil-induced in-
jury in the liver than expression on venular endothelium.
Two important questions remain. 1) Why is there only neutro-
phil accumulation in the liver vasculature despite the fact that other
leukocytes, e.g., lymphocytes and monocytes, also express LFA-1
and VLA,? An infiltration of mononuclear cells in the liver has
been described 24 to 48 h after ET administration (50), and the
accumulation of monocytes in the liver several days after treatment
with
Propionibacrerium
acnes
could be inhibited by Abs to LFA-1
(5
I,
52) and ICAM-1 (52). However, in the GalET model, severe
neutrophil-induced injury followed by hepatic failure and shock
develop within
7
to
10
h (19), i.e., at a time when few mononuclear
cells are recruited into the liver. A similar temporal sequence of
neutrophil and monocyte accumulation was observed in a perito-
nitis experiment (12). These observations suggest that expression
of adhesion molecules on sinusoidal lining cells is necessary for
leukocyte sequestration and transmigration; however, by them-
selves they are not sufficient. Generation of leukocyte-specific
chemotactic factors, e.g., chemokines, may play a role in the tem-
poral sequence of leukocyte recruitment to an inflammatory site. 2)
What is the role of Gal in this model? Gal cotreatment does not
affect cytokine formation (21), NF-KB activation in all liver cells
(37,
38),
the up-regulation
of
ICAM-1 (21) or VCAM-1 (Fig. 5) in
the hepatic vasculature, and the sequestration of neutrophils (21,
49). However, Gal cotreatment drastically increases the number of
extravasating neutrophils and liver injury (49). This observation
led to the hypothesis that parenchymal cells may generate chemo-
tactic signals in GaVET-treated mice, in which neutrophils trans-
migrate and cause injury, compared with ET-treated animals, in
which neutrophils remain sequestered in sinusoids without causing
liver damage (49). One possible explanation for this difference
may be the formation of neutrophil chemoattractant C-X-C che-
mokines in hepatocytes (53-55). Indeed, substantially higher for-
mation of KC/Gro, a murine member
of
the C-X-C chemokine
family, was detected in GaVET-treated mice than in those given
ET treatment alone (56). An alternative signal could be generated
by the substantial number of hepatocytes undergoing apoptosis in
the GaVET group at the time when neutrophils begin to extravasate
(49). DNA fragmentation and apoptotic cell death are only ob-
served with Gal/ET and not with ET treatment alone (57). We are
currently investigating these different hypotheses.
In summary, our data demonstrated a cytokine-dependent acti-
vation of VCAM-1 gene transcription in the liver during endotox-
emia in vivo. Increased levels
of
VCAM-1 mRNA and VCAM-1
protein expression are restricted to endothelial cells and Kupffer
cells and could not be detected on hepatocytes. A mAb to
VCAM-
1
significantly attenuated liver injury in Gal/ET-treated
Downloaded from http://journals.aai.org/jimmunol/article-pdf/158/12/5941/1075894/5941.pdf by guest on 23 December 2022
The
Journal
of
Immunology
5947
animals. The
Ab
had no effect
on
neutrophil accumulation in he-
patic sinusoids, but reduced transendothelial migration and, there-
fore, prevented attack on parenchymal cells. These results suggest
that neutrophils are able to use
VLA-4NCAM-1
interactions for
transmigration
in
the liver vasculature.
Acknowledgments
We
thank Dr. Anthony
M.
Manning (Cell Biology and Inflammation, Phar-
macia
&
Upjohn,
Inc.)
for
providing
mouse
and rat
VCAM-1
cDNA and
Greg
E.
Winterrowd
(Cell
Biology
and
Inflammation, Pharmacia
&
Up-
john,
Inc.)
for
technical assistance with
flow
cytometry.
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