Platelet
endothelial cell adhesion molecule-1 (PECAM-1) is thought to be
critical for transendothelial migration of leukocytes, including
neutrophils. Because neutrophil-mediated liver injury during
endotoxemia is dependent on transmigration, we investigated the role of
PECAM-1 in the pathophysiology of endotoxin-induced liver
injury. Male C3Heb/FeJ mice were treated with
galactosamine (Gal) and endotoxin (ET) (700 mg/kg Gal/100 µg/kg ET),
and liver sections were stained for PECAM-1 expression. Control livers
showed the presence of PECAM-1 on endothelial cells of large vessels but not in sinusoids. Gal/ET treatment did not change the expression pattern of PECAM-1. Gal/ET-induced liver injury (area of necrosis: 38 ± 3%) was not attenuated by treatment with 3 mg/kg of the
antimurine PECAM-1 antibody 2H8. The antibody had no effect on
sequestration and transmigration of neutrophils in sinusoids or the
margination of neutrophils in large vessels. In contrast, 2H8 inhibited
glycogen-induced neutrophil migration into the peritoneum by 74%; this
effect correlated with PECAM-1 expression in the intestinal
vasculature. Thus PECAM-1 is neither expressed nor inducible in hepatic
sinusoids and is consequently not involved in neutrophil transmigration
in the liver during endotoxemia. On the other hand, expression of
PECAM-1 in mesenteric veins is critical for peritoneal neutrophil
accumulation.
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INTRODUCTION |
PLATELET-ENDOTHELIAL CELL adhesion molecule-1
(PECAM-1; CD31) is a member of the immunoglobulin gene
superfamily (30, 33, 34). PECAM-1 is expressed on leukocytes, i.e.,
platelets, neutrophils, monocytes, and selected T cell subsets (5, 29).
On endothelial cells, PECAM-1 is concentrated at the intercellular
junction (5, 29). Pretreatment of monocytes or neutrophils with an
antibody against PECAM-1 prevented leukocyte transmigration across an
endothelial cell monolayer without inhibition of adherence (26). On the other hand, treatment of endothelial cells with the PECAM-1 antibody was also effective in inhibiting leukocyte transmigration. These data
suggest that PECAM-1 on leukocytes and on endothelial cells is
critically involved in the transendothelial migration process (26).
Recently, these in vitro findings have been substantiated in a variety
of in vivo models. Anti-PECAM-1 antibodies attenuated neutrophil
accumulation in the alveolar space in an IgG immune complex model of
lung inflammation (36) and in the peritoneum after glycogen or
thioglycolate administration (2, 36). Furthermore, neutrophil
transmigration and tissue necrosis was inhibited by anti-PECAM-1
antibodies without affecting overall neutrophil accumulation in a
feline model of myocardial ischemia-reperfusion (I/R) injury (27). In a related study using a rat model of myocardial infarction, Fab2 fragments of PECAM-1
antibodies attenuated tissue necrosis and reduced neutrophil
infiltration into the necrotic tissue (12). However, the role of
PECAM-1 has not been investigated in any experimental model of liver
inflammation.
Neutrophils contribute to liver injury during hepatic I/R (17, 19),
endotoxemia (13, 20), alcoholic intoxication (1, 25), hepatic chemokine
overexpression (24), partial hepatectomy (4), or intestinal I/R (14,
15). General mechanisms of neutrophil-induced liver injury include the
sequestration of these cells in the hepatic vasculature,
transmigration, and adherence to parenchymal cells (22). Similar to
many other organs (11), e.g., intestine, heart, skeletal muscle, and
skin, neutrophil margination takes place in hepatic postsinusoidal
venules (3, 37). However, most neutrophils sequestered in the liver
vasculature are located in sinusoids; neutrophils that migrate out of
the sinusoids have been shown to be responsible for parenchymal cell injury in an endotoxin (ET) shock model (3). The importance of the
transendothelial migration step for the pathophysiology was further
demonstrated by antibodies against intercellular adhesion molecule-1
(ICAM-1) (7) and vascular cell adhesion molecule-1 (VCAM-1) (6). These
antibodies protected against liver injury by inhibiting the
extravasation of sinusoidal neutrophils. Because of the critical role
of PECAM-1 in leukocyte transendothelial migration, as demonstrated in
the lung, intestine, skin, and heart (2, 12, 27, 36), we evaluated the
expression of PECAM-1 in the hepatic vasculature and characterized the
effect of an anti-PECAM-1 antibody on hepatic neutrophil sequestration,
transendothelial migration, and parenchymal cell injury in a
well-established model of ET-induced liver failure. For comparison, we
investigated PECAM-1 expression in mesenteric vessels and the effect of
the anti-PECAM-1 antibody in the glycogen peritonitis
model.
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MATERIALS AND METHODS |
Animals.
Male mice (strain C3Heb/FeJ; 20-25 g body wt) were purchased
from Jackson Laboratories (Bar Harbor, ME). The animals had free access
to food (Certified Rodent Diet #5002C; PMI Feeds, Richmond, IN) and
water. The experimental protocols followed the criteria of Pharmacia & Upjohn, Inc., and the National Institutes of Health Guide for the Care and Use of Laboratory
Animals. Animals were treated intraperitoneally with
700 mg/kg D-galactosamine (Gal; Sigma, St. Louis, MO) and 100 µg/kg Salmonella
abortus equi-ET (Sigma) dissolved in sterile PBS (pH
7.0). Some animals were treated with 3 mg/kg anti-mouse PECAM-1
antibody (clone 2H8) (Endogen, Cambridge, MA), control IgG (Sigma), or
anti-mouse P-selectin antibody (clone RB40.34) (PharMingen, San Diego,
CA) intravenously at the time of Gal/ET injection.
Experimental protocols.
The animals were killed by cervical dislocation 4 or 7 h after
administration of Gal/ET. Blood was collected from the right ventricle
into a heparinized syringe and centrifuged, and plasma was used for
determination of alanine aminotransferase (ALT) activity with Sigma test kit DG 159-UV. Pieces of the liver or the mesentery were fixed in phosphate-buffered Formalin for histological analysis or
embedded in Tissue-Tek O.C.T./Sakura Finetek, Torrance,
CA) embedding medium and snap-frozen in liquid
nitrogen-chilled methylbutane for immunohistochemistry.
Histology.
Formalin-fixed portions of the liver were embedded in paraffin,
and sections 5 µm thick were cut. Neutrophils were stained with the
use of the AS-D chloroacetate esterase technique as
described in detail previously (18). Neutrophils were identified by
positive staining and morphology and were counted in 50 high-power
fields (magnification, ×400), using a Nikon Labophot microscope.
Only neutrophils present within sinusoids or extravasated into the tissue were counted; the number of neutrophils marginated within large
vessels, e.g., hepatic veins, was evaluated separately. Cell damage was
evaluated in parallel sections stained with hematoxylin and eosin. We
estimated the percentage of cell necrosis by evaluating the number of
microscopic fields with necrosis compared with the entire histological
section. The pathologist (A. Farhood) performing the histological
evaluation (number of neutrophils, area of necrosis) was
blinded as to the treatment of animals.
Immunohistochemistry.
Cryostat sections (8 µm) of livers and mesentery were
dehydrated in acetone, air-dried, and fixed in 2% buffered Formalin (2 min). For localization of PECAM-1 (CD31) or ICAM-1 (CD54), slides were
washed in PBS buffer, labeled with rat anti-murine CD31 antibody (MEC
13.3, PharMingen), rat anti-murine CD54 (YN1/1.7.4, American Type
Culture Collection, Rockville, MD), or an appropriate negative antibody
control (R35-95, PharMingen) at 3.0 µg/ml for 30 min. Sections
were washed in buffer, and endogenous peroxidase activity was reduced
by dehydrating the tissues with methanol containing 3% hydrogen
peroxide for 20 min. Slides were rehydrated with buffer and labeled
with 25 µg/ml of peroxidase-conjugated mouse anti-rat IgG secondary
antibody Fab2 fragments (Jackson ImmunoResearch, West Grove, PA) for 30 min. Secondary antibody was
detected by using 3,3'-diaminobenzidine tetrahydrochloride tablets (Sigma) in 50 mM Tris · HCl and 150 mM sodium
chloride (pH 7.6) for 10 min. Sections were washed and counterstained
with Mayer's hematoxylin solution (Sigma) for 3 min.
Peritonitis experiments.
To test the efficacy of the anti-PECAM-1 antibody to inhibit adhesion
to vascular endothelium and transendothelial migration, animals were
intravenously injected with 250 µl of saline, 3 mg/kg of clone 2H8,
or control immunoglobulin. At the same time, the animals
received an intraperitoneal injection of saline (500 µl) or glycogen
(1 g/kg body wt) in sterile saline. After 4 h, the animals were killed
and their peritoneal cavities were lavaged twice with 2 ml of PBS. The
lavage fluids were centrifuged (1,000 g) for 10 min to sediment the
neutrophils. The pellets were resuspended in Tris-buffered 0.75%
NH4Cl for 10 min to lyse
erythrocytes. After centrifugation, the pellets were resuspended in
detergent buffer (50 mM phosphate buffer containing 0.5%
cetyltrimethylammonium bromide), briefly sonicated, and freeze-thawed
twice. Myeloperoxidase (MPO) activity as an index for neutrophil
accumulation was determined spectrophotometrically in 50 mM phosphate
buffer (pH 6.0) containing 0.165 mg/ml
o-dianisidine hydrochloride and 0.15 mM hydrogen peroxide (23). The change in absorbance was determined at
460 nm. In a separate experiment, the cell pellet was resuspended in
HEPES buffer and stained with modified Giemsa-Wright stain (Neat Stain; Midlantic Biomedical, Paulsboro, NJ) for differential cell count.
Statistics.
All data are means ± SE. Statistical significance between the
control group and a treated group was determined with the unpaired Student's t-test or Wilcoxon's
rank-sum test. Comparisons between multiple groups were performed with
one-way ANOVA followed by Bonferroni
t-test.
P < 0.05 was considered
significant.
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RESULTS |
Administration of Gal/ET caused severe liver injury at 7 h as indicated
by high plasma ALT values and substantial hepatocellular necrosis (Fig.
1). Preceding liver injury (4 h), large numbers of
neutrophils accumulated in sinusoids (Fig. 2). At the
time of injury (7 h), there was a moderate further increase in the number of neutrophils (Fig. 2); ~30-35% of these leukocytes had transmigrated by that time. In addition to sinusoidal
sequestration of neutrophils, there was margination of neutrophils in
postsinusoidal venules at 4 h (Fig. 3). However, at 7 h
no neutrophils were observed in the lumen of larger vessels or in the
surrounding tissue (data not shown). Treatment with an anti-mouse
PECAM-1 antibody (3 mg/kg) did not significantly change
plasma ALT activity or the area of necrosis after Gal/ET administration
compared with control IgG-treated or untreated animals (Fig. 1). The
anti-PECAM-1 antibody had no effect on hepatic neutrophil sequestration
in sinusoids before liver injury (4 h) or at the time of injury (7 h)
(Fig. 2). Furthermore, the PECAM-1 antibody did not affect neutrophil
margination in postsinusoidal venules at 4 h (Fig. 3). As a control
experiment, the anti-P-selectin antibody RB40.34 attenuated neutrophil
margination in these large vessels by 76% at 4 h after Gal/ET (Fig. 3)
but had no effect on sinusoidal neutrophil sequestration (data not shown).

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Fig. 1.
Liver injury, as assessed by plasma alanine aminotransferase (ALT)
activity and histologically by the area of necrosis, was evaluated in
C3Heb/FeJ mice 7 h after combined administration of 700 mg/kg
galactosamine (Gal; G) and 100 µg/kg Salmonella
abortus equi-endotoxin (ET). Animals were pretreated
with 3 mg/kg murine anti-platelet endothelial cell adhesion molecule-1
(anti-PECAM-1) antibody (clone 2H8) or isotype-matched control IgG. C,
control. Data are means ± SE of
n = 8 animals per group. There is no
statistically significant difference between groups. For comparison,
ALT values for untreated control animals were 21 ± 4 U/l with 0%
necrosis.
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Fig. 2.
Hepatic neutrophil sequestration was evaluated in C3Heb/FeJ mice
4 and 7 h after combined administration of 700 mg/kg Gal and 100 µg/kg Salmonella abortus ET. Animals
were pretreated with 3 mg/kg murine anti-PECAM-1 antibody (clone 2H8)
or isotype-matched control IgG. Neutrophils were counted in 50 high-power fields (HPF). PMN, polymorphonuclear leukocytes
(neutrophils). Data are means ± SE of
n = 5 animals (4 h) or
n = 8 animals (7 h) per group.
* P < 0.05 compared with
untreated controls (C).
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Fig. 3.
Neutrophil margination in large hepatic vessels, i.e., portal and
postsinusoidal venules, was evaluated 4 h after combined administration
of 700 mg/kg Gal and 100 µg/kg Salmonella
abortus ET. Animals were treated with 3 mg/kg murine
anti-PECAM-1 antibody (2H8), control IgG, or anti-P-selectin antibody
(clone RB40.34). No marginated PMN were found in these vessels in
control livers or 7 h after Gal/ET. Neutrophils were counted in 10 large vessels of approximately similar size. Data are means ± SE of
n = 5 animals per group.
* P < 0.05 compared with
untreated controls (C). # P < 0.05 compared with IgG.
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To assess the expression of PECAM-1 in the hepatic vasculature,
immunohistochemical analysis for PECAM-1 was performed.
In control animals, there was clear PECAM-1 expression on large vessel endothelial cells; however, no PECAM-1 could be detected on sinusoidal lining cells (Fig. 4). The expression pattern of PECAM-1
in ET-treated animals (4 h) was identical to that observed in control
livers. Thus PECAM-1 appears to be constitutively expressed selectively on large-vessel endothelial cells. For comparison, sections from the
same livers were stained with an anti-ICAM-1 antibody (Fig. 4). In
control animals, ICAM-1 was moderately expressed on large vessel
endothelium and lightly expressed on sinusoidal lining cells. However,
4 h after ET, ICAM-1 expression was substantially increased on all
endothelial cells, particularly in sinusoids.

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Fig. 4.
Immunohistochemical staining of control or Gal/ET-challenged mice
livers for intercellular adhesion molecule-1 (ICAM-1) or PECAM-1.
a: Rat anti-mouse ICAM-1 staining of
normal mouse liver. Brown color indicates positive staining (arrow).
Note strong vascular labeling with some sinusoidal staining.
Counterstain for cell nuclei: Mayer's hematoxylin.
b: Rat anti-mouse ICAM-1 staining in
Gal/ET-treated liver. Note strong staining of central vein (cv) and
surrounding sinusoids. c: Rat
anti-mouse PECAM-1 staining of normal liver. Arrows indicate positive
staining on large and small vessels but no staining of sinusoids.
d: PECAM-1 staining of a
Gal/ET-challenged mouse liver gave a staining pattern similar to that
seen in control animals. Original magnification, ×400.
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To test the efficacy of the anti-PECAM-1 antibody batch and the dose
used, glycogen peritonitis experiments were performed. This model is
useful to assess the effect of therapeutic interventions on neutrophil
adhesion and transmigration in mesenteric vessels in vivo (18).
Intraperitoneal injection of glycogen induced substantial neutrophil
infiltration within 4 h, as indicated by high MPO activity in the
peritoneal lavage fluid (Fig. 5). The anti-PECAM-1
antibody (3 mg/kg) inhibited MPO activity by 74% compared with control
animals or control IgG-treated animals. Immunohistochemical evaluation
of PECAM-1 expression showed strong staining in both intestinal
arteries and veins in control animals (Fig. 6). This
expression pattern and its intensity did not change during glycogen
peritonitis (Fig. 7). However, numerous leukocytes could
be detected in the mesenteric veins during the inflammatory response
(Fig. 7a); staining with the GR-1
antibody (Fig. 7b) identified these
leukocytes as granulocytes. For comparison, parallel sections were
stained for ICAM-1 (Figs. 6c and
7c); in contrast to PECAM-1, ICAM-1
was predominantly expressed on venular endothelium.

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Fig. 5.
Myeloperoxidase (MPO) activity in peritoneal lavage fluid as an index
of neutrophil infiltration 4 h after intraperitoneal injection of 1 ml/kg saline (controls) or 1 g/kg glycogen (Glc) in sterile saline.
Some animals were pretreated with 3 mg/kg murine anti-PECAM-1 antibody
(clone 2H8) or isotype-matched control IgG. Data are means ± SE of n = 5 animals per group.
* P < 0.05 (controls vs.
GLC-treated animals). # P < 0.05 compared with Glc or Glc/IgG.
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