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THE ROLE OF P-SELECTIN AND ITRACELLULAR ADHESION
MOLECULE-1 IN HEPATIC ISCHEMIA/REPERFUSION INJURY

presented by

Curtis Steven Young

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of the requirements for

Masters Sur e
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W chr

Elahé Crockett, Ph.D.

 

 

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6/01 cJCIRC/DateDuthS‘pJS

THE ROLE OF P-SELECTIN AND INTERCELLULAR ADHESION MOLECULE-1
IN HEPATIC lSCHEMIA/REPERFUSION INJURY

BY

Curtis Steven Young

A THESIS

Submitted to
Michigan State University
in partial fulﬁllment of the requirements
for the degree of

MASTERS OF SURGERY

Department of Surgery

2001

ABSTRACT

THE ROLE OF P-SELECTIN AND INTERCELLULAR ADHESION MOLECULE-1
IN HEPATIC lSCHEMIA/REPERFUSION INJURY

By

Curtis Steven Young

Neutrophil recruitment and emigration represent one of the early cellular
events that occur following hepatic ischemia/reperfusion (IR) injury. These two
factors play a critical role in determining the extent of hepatocellular damage.
Adhesion molecules on neutrophils and endothelial cells regulate the migration of
neutrophils from the vasculature to the sites of injury. The goal of this research
was to evaluate the role of the adhesion molecules P-selectin and intercellular
adhesion molecule-1 (lCAM-1) in neutrophil-mediated hepatic IR injury utilizing
P-selectin/ICAM-1 double knockout (P/l null) mice.

Male wild-type C578L/6 and I’ll null mice underwent a midline Iaparotomy
with 90 minutes of lobar hepatic ischemia followed by reperfusion at various time
points. Liver injury was determined by changes of plasma alanine
aminotransferase (ALT) levels and histopathology. Immunohistochemical
staining of hepatic tissue sections was prepared to assess lCAM-1 expression
and neutrophil recruitment.

The results demonstrated that in both wild-type and PH null mice,
reperfusion following ischemia induced signiﬁcant hepatocellular injury. The

injury was minimal in both sham operated mice and at 0 hours of reperfusion.

lmmunohistochemistry of lCAM-1 conﬁrmed the absence of expression in the PH
null mice, as well as constitutive and inducible expression of lCAM-1 in wild-type
mice. Histopathology and immunohistochemical staining revealed recruitment
of neutrophils into the hepatic parenchyma after IR in both wild-type and P/l null
mice. There was no signiﬁcant difference in hepatocellular injury in response to
IR between wild-type and P/l null mice. This investigation suggests that P-
selectin and lCAM-1 do not play a critical role in neutrophil recruitment and

hepatocellular injury following IR.

Copyright by
Curtis Steven Young
2001

I would like to dedicate this thesis to my daughters, Elizabeth and Amanda, who
lovingly remind me of why I work so hard in the ﬁrst place.

ACKNOWLEDGMENTS

First, I would like to express my deepest gratitude to Dr. Richard E. Dean,
MD, Professor and Chairman of the Department of Surgery forthe opportunity
to become a surgical resident here at Michigan State University and the privilege
to pursue basic science research. His dedication to the residency program and
research experience has provided a nurturing environment that has fostered my
development as both a surgeon and scientist.

I would also like to express my gratitude to my major professor, Dr. Elahe
Crockett, Ph.D., Director of Basic Science Research Program and the Chair of
my Research Thesis Committee, for her academic counseling, advice,
encouragement, as well as assistance throughout the course of this study.

I would especially like to thank Dr. Alan Davis, Ph.D., for his invaluable
advice, support and assistance with this research project, and statistical analysis.
I am particularly grateful for his insight and approach to research.

I would like to acknowledge the other members of my graduate committee:
Dr. Douglas F. Naylor, MD, for his advice and dedication to teaching; Dr. L. Rao
Kareti, MD, for his advice and technical expertise; and Dr. Jack Harkema,
D.V.M., Ph.D., for his teaching and assistance with histopathologic examination
of tissues, as well as his critique of the manuscript.

I owe a world of thanks to Crystal Remelius, 8.8., Research Assistant,
and Karen Hess, M.S., Research Assistant for their technical assistance, support

and patience.

vi

I also owe my deepest thanks to my friend and colleague, Dr. Juan Palma,
MD, for all of his contributions to this project. He was an indispensable
resource for his technical expertise in the animal model, as well as his insight into
this area of research.

To my dear friend and colleague, Dr. Ben Mosher, MD, I thank him for
laying the foundation for this project. I am also thankful for his encouragement
and for being the standard to which I compared myself, but could never achieve.

Finally, I would like to thank my wife, Carmela, for her support and

dedication.

vii

TABLE OF CONTENTS

LIST OF TABLES .................................................................................. ix
LIST OF FIGURES ................................................................................. x
LIST OF ABBREVIATIONS ..................................................................... xi
INTRODUCTION ................................................................................... 1
REVIEW OF THE LITERATURE ............................................................... 5
Hepatic lschemia/Reperfusion Injury ................................................. 5

Early Phase ........................................................................ 5

Late Phase ....................................................................... 1O

lntercellular Adhesion Molecules .................................................... 1 1
Regulation of Hepatic Adhesion Molecule Expression ......................... 13
Adhesion Molecules and Neutrophil-Induced Liver Damage ................. 17

Adhesion Molecule Deﬁcient Mice in Models of Ishemia/Reperfusion.....20
P-selectin/lCAM-I Double Mutant Mice in Models of Inﬂammation.........29

MATERIALS AND METHODS ................................................................ 31

RESULTS .......................................................................................... 40
DISCUSSION ...................................................................................... 52
REFERENCES .................................................................................... 65

viii

LIST OF TABLES

TABLE 1: Summary of Plasma ALT Levels in Wild-Type and P/l Null
Mice After Sham Operation and IR ....................................... 44

FIGURE 1:
FIGURE 2:

FIGURE 3:

FIGURE 4:

FIGURE 5:

LIST OF FIGURES

Model of Partial Hepatic lschemia ....................................... 34
Veriﬁcation of ICAM-1 Deﬁciency in P/I Null Mice .................... 41

Hepatocellular Injury After 90 Minutes of Hepatic lschemia With
Various Reperfusion Times — Wild-Type vs. P/l Null Mice ......... 45

Demonstration of Hepatocellular Injury by Histopathology —
Hematoxylin and Eosin Staining .......................................... 47

Demonstration of Neutrophil Recruitment by
Immunohistochemical Staining ............................................ 50

LIST OF ABBREVIATIONS

Adenosine Triphosphate .................................................................................. ATP
Alanine Aminotransferase ................................................................................ ALT
Aspartate Aminotransferase ............................................................................. AST
Cytokine-lnduced Neutrophil Chemoattractant .............................................. CINC
Epithelial Neutrophil Activating Protein-78 ................................................. ENA-78
lntercellular Adhesion Molecule-1 ............................................................... lCAM-1
Interleukin-1 ..................................................................................................... IL-1
Interleukin-6 ..................................................................................................... lL-6
Interleukin-8 ..................................................................................................... lL-8
lschemia/Reperfusion ......................................................................................... IR
Lymphocyte Function-Associated Antigen-1 ................................................. LFA-1
Macrophage-1 Antigen .................................................................................. Mac-1
Macrophage Inﬂammatory Protein-2 ............................................................. MIP-2
Messenger Ribonucleic Acid ........................................................................ mRNA
Monoclonal Antibody ....................................................................................... mAb
Nitric Oxide ....................................................................................................... NO
Nuclear Factor KB ........................................................................................... NFKB
P-selectin/lCAM-1 Double Knockout ........................................................... P/I Null
PIatelet-Endothelial Cell Adhesion Molecule-1 ........................................ PECAM-1
P-selectin Glycoprotein Ligand-1 ............................................................... PSGL-1
Reactive Oxygen Species ............................................................................... ROS
Standard Error of Mean ................................................................................... SEM

xi

LIST OF ABBREVIATIONS

Tumor Necrosis Factor on .............................................................................. TNFa
Vascular Cell Adhesion Molecule-1 ........................................................... VCAM-1
Very Late Antigen ............................................................................................. VLA

xii

INTRODUCTION

The functional consequences of depriving a tissue of its blood supply have
been appreciated for many years. Recently, however, it has become evident that
reperfusion, the restoration of blood ﬂow after a period of ischemia, can place
ischemic organs at risk of further cellular necrosis and thereby limit functional
recovery (Carden and Granger, 2000). Much of the current understanding of
ischemia/reperfusion (IR) injury is based on studies from many different organs,
including brain, heart, lung, kidney, liver, intestine and skeletal muscle.
Reperfusion of ischemic tissues results in an acute inﬂammatory response, which
is characterized by an increased ﬂuid ﬁltration and steric hindrance of circulating
blood cells in capillaries, with a concomitant recruitment of adherent and
emigrating leukocytes. The results of several investigations suggest that there is
a cause-effect relationship between the accumulation of inﬂammatory cells and
the microvascular, as well as parenchymal cell, dysfunction that are elicited by IR
(Oliver, 1991; Horie et al., 1997).

Compromised blood ﬂow to the liver, followed by resumption of normal
blood ﬂow causes hepatic damage known as IR injury. Hepatic IR injury is an
important clinical problem that often follows circulatory shock with resuscitation,
as well as many types of liver surgery. Total interruption of hepatic blood ﬂow is
sometimes necessary during repair of liver injury or during resection for tumors
(Feliciano et al., 1986; Pachter and Spencer, 1979). Furthermore, the liver is
subjected to both warm and cold ischemia during hepatic transplantation

(Toledo-Pereyra, 1991 ). It is well established that the liver is quite sensitive to

ischemia, and morbidity and mortality Increase rapidly with lengthening of the
ischemic interval (Chien et al., 1977). Interruption of blood ﬂow to the liver can
result in cellular injury from the ischemia itself, but there is an additional
component of injury that occurs during restoration of blood ﬂow. Therefore, while
reperfusion is essential to prevent the progression of metabolic and structural
abnormalities during hepatic ischemia, paradoxically it can increase the degree
of injury. The clinical consequences of hepatic IR injury include liver failure and
multiple organ system failure, both of which carry high rates of morbidity and
mortality (Faist et al., 1983; Keller et al., 1985).

Although the sequelae of hepatic IR injury are well known, the exact
mechanisms remain elusive. Several mechanisms have been proposed
including enhanced production of proinﬂammatory mediators (Camargo et al.,
1997; Clavien 1997; Jaeschke et al., 1991), recruitment of polymorphonuclear
leukocytes (Jaeschke et al., 1990; Del Zoppo et al., 1991; Suzuki et al., 1993)
and occlusion of ﬂow (Ames et al., 1968; Koo et al., 1992).

A few studies have described hepatic IR injury as being comprised of two
phases (Jaeschke et al., 1990). The early phase develops during the ﬁrst one to
three hours of reperfusion. It is characterized by increased levels of liver
transaminases, both alanine aminotransferase and aspartate aminotransferase.
This phase is mediated through a reactive oxygen species (ROS) mechanism
and correlates with the activation of Kupffer cells. The injury incurred during this
phase is independent of intrahepatic neutrophils (Jaeschke and Farhood, 1991;

Jaeschke et al., 1992). The late phase occurs after six to twenty-four hours of

reperfusion. During this phase there is further increase in transaminases and
massive emigration of neutrophils into the hepatic parenchyma (Jaeschke et al.,
1993; Langdale et al., 1993; Suzuki et al., 1993).

Neutrophil recruitment and emigration represent one of the early cellular
events that occur following hepatic IR injury and play a critical role in determining
the extent of hepatocellular damage. Adhesion molecules on neutrophils and
endothelial cells regulate the migration of neutrophils from the vasculature to the
sites of inﬂammation. The hypothesis of this study is that the cellular adhesion
molecules P-selectin and intercellular adhesion molecule-1 (ICAM-1) play a
pivotal role in neutrophil-mediated hepatic IR injury. The goal of this research
was to evaluate the role of the adhesion molecules P-selectin and lCAM-1 in
neutrophil-mediated hepatic IR injury utilizing P-selectin/lCAM-1 double knockout
(P/l null) mice.

Male wild-type C57BL/6 and PH null mice underwent a midline Iaparotomy
with 90 minutes of lobar hepatic ischemia followed by reperfusion at various time
points. Liver injury was determined by changes of plasma alanine
aminotransferase (ALT) levels and histopathology. Immunohistochemical
staining of hepatic tissue sections were prepared to assess lCAM-1 expression
and neutrophil recruitment.

Signiﬁcant hepatocellular injury was induced in a model of warm hepatic
ischemia/reperfusion injury in both wild-type and P/I null mice. Minimal injury
was incurred in both sham operated mice and mice that underwent ischemia and

0 hours of reperfusion. lmmunohistochemistry for ICAM-1 conﬁrmed the

absence of expression in the PH null mice, as well as constitutive and inducible
expression of lCAM-1 in wild-type mice. Signiﬁcant neutrophil inﬁltration into
the hepatic parenchyma after IR was identiﬁed on immunohistochemical staining
in both wild-type and P/l null mice. However qualitatively, there was less
neutrophil inﬁltration in the livers of PH null mice. Hepatocellular injury, as
assessed by serum ALT levels, was not signiﬁcantly different between wild-type
and PH null mice. Collectively, the results of this investigation suggest that P-
selectin and ICAM-1 do not play a critical role in neutrophil recruitment and

hepatocellular injury following IR.

REVIEW OF THE LITERATURE

Hepatic lschemia/Reperfusion Injury: Early Phase

As the cells become hypoxic, they shift from aerobic to anaerobic
metabolism, which results in derangements in the intracellular milieu. Anaerobic
glycolysis leads to increased lactic acid production and accumulation, with
subsequent lowering of the intracellular pH. Hydrolysis of adenosine
triphosphate (ATP) further exacerbates the decrease in pH. This naturally
occurring acidosis protects against the onset of necrotic cell death in hepatocytes
and other cells (Bonventure and Cheung, 1985; Gores et al., 1988; Gores et al.,
1989). However, the recovery of normal a pH after reperfusion of ischemic cells
then accelerates cell killing, a phenomenon known as the pH paradox (Gores et
al., 1988; Currin et al., 1991; Bond et al., 1991; Bond et al., 1993; Harper et al.,
1993; Zager et al., 1993; Kaplan et al., 1995; Currin et al., 1996; Qian et al.,
1997; Nishimura et al., 1998; Lichtman and Lemasters, 1999). Treatments that
delay the recovery of intracellular pH after reperfusion, such as Na‘lH” exchange
inhibitors and Na+-free reperfusion, prevent pH dependent reperfusion injury
(Lichtman and Lemasters1999). However, accelerated recovery of intracellular
pH by substances such as monensin, hastens cell death after reperfusion (Bond
et al., 1993; Harper et al., 1993; Zager et al., 1993; Kaplan et al., 1995; Currin et
al., 1996; Qian et al., 1997; Nishimura et al., 1998; Lichtman and Lemasters
1999). Cytoprotection by an acidic intracellular pH appears to be partially
mediated by suppression of degradative enzymes such as proteases and

phospholipases that are suppressed by an acidic pH but are activated at normal

pH during reperfusion (Harrison et al., 1991; Bronk and Gores, 1993; Arora et al.,
1996; Harrison-Shostak et al.,1997).

Another consequence of hepatic IR in the early phase of reperfusion injury
is the activation of Kupffer cells (Jaeschke and Farhood, 1991; Lindert et al.,
1992). Activated Kupffer cells release tumor necrosis factor a (TNFa), interleukin
1 (IL-1) and ROS, which aggravate and escalate the process of hepatic IR injury
(Lichtman et al., 1994). Kupffer cells primed in the early phase worsen IR injury
(Shiratori et al., 1994), whereas inactivation of Kupffer cells with methyl palmitate
or gadolinium chloride protects against warm hepatic IR injury and decreases
oxidative stress (Jaeschke and Farhood, 1991; Bremer et al., 1994).

A signiﬁcant consequence of Kupffer cell activation is the release of TNFa,
which has local as well as systemic effects. Tumor necrosis factor or released
after IR is derived from the liver and induces the release of epithelial neutrophil
activating protein-78 (ENA-78), which is a powerful chemotactic agent for
neutrophils (Muraoka et al., 1997).

Overall, the evidence that TNFa plays a role in hepatic IR injury is
substantial. The precise stimulus for its release by Kupffer cells remains poorly
understood but may involve anoxia, perturbations in blood ﬂow and ROS.
Despite the experimental studies indicating a primary role for TNFa in IR injury,
clinical studies have not supported these observations (Chazouilleres et al.,
1992, Clavien et al., 1996).

In addition to TNFa, lL-1 and lL-6 have been shown to be involved in the

pathophysiology of hepatic IR injury. In vivo lL-6 release is delayed compared to

TNFa and lL-1, which increase within minutes of IR injury (Wanner et al., 1996).
Interleukin-1 release is elevated in rat Kupffer cells that have been isolated after
reperfusion (Wanner et al., 1996). Interleukin-1 promotes ROS production
(Shirasugi et al., 1997), whereas an lL-1 receptor antagonist attenuates hepatic
IR injury in rat and lowers TNFa levels, suggesting that IL-1 promotes TNFa
production (Shito et al., 1997). Treatment with lL-6 protects against warm
hepatic IR injury in rats (Camargo et al., 1997). Yet other studies demonstrate
that hypoxia induces the release of lL-6 from cultured endothelial cells and
lymphocytes through activation of nuclear factor KB (NFKB), an effect not
mediated by reperfusion (Muraoka et al., 1997).

Epithelial neutrophil activating protein-78 is a member of a group of
molecules known as chemotactic cytokines, or chemokines. These
Chemoattractant proteins have a varying speciﬁcities and activities on different
leukocytes. Other members of the chemokine family include IL-8, platelet factor
4, cytokine-induced neutrophil Chemoattractant (CINC), and macrophage
inﬂammatory protein (MlP)—2 (Lichtman and Lemasters, 1999). Cytokine-
induced neutrophil chemattractant is another important chemokine released from
activated Kupffer cells. It is released within hours of IR injury and also acts as a
potent Chemoattractant for neutrophils (Lichtman and Lemasters, 1999).
Cytokine-induced neutrophil chemattractant links the early phase of hepatic IR
injury mediated my Kupffer cells with the late phase of injury mediated by
neutrophils (Lichtman and Lemasters, 1999). Macrophage inﬂammatory protein-

2 is another chemokine involved in hepatic IR injury. Its mRNA begins to

increase within 3 hours of IR injury and continues to rise for at least 9 hours
(Lentsch et al., 1998); both MlP—2 and CINC increase integrin expression on
neutrophils, which further promotes transmigration from the microvasculature
(Shanley et al., 1997).

The hepatocyte is susceptible to injury by ROS. The mechanisms of
oxygen-derived free radical formation include activation of phospholipase and the
arachidonic acid cascade, as well as activation of Kupffer cells and neutrophils.
These metabolites may also result from the breakdown products of ATP and the
oxidative stress secondary to the reduction of glutathione during reperfusion
(Atalla et al., 1985; Metzger and Lauterburg, 1988). Sources of ROS include
endothelial cells, Kupffer cells, neutrophils and hepatocytes (Dahm et al., 1991;
Drath and Karnovsky, 1975). Kupffer cells are a main source of ROS in the early
phase of hepatic IR injury (Caldwell-Kenkel 1991; Jaeschke 1991; Jaeschke
1992), whereas neutrophils are the major source of ROS during the later phase
of injury (Atalla et al., 1985; Marabayashi et al., 1986).

These alterations disrupt enzyme systems that maintain normal cellular
homeostasis. During this time, xanthine dehydrogenase is irreversibly converted
to xanthine oxidase via a calcium-dependent protease (Batelli 1980; Della Corte
and Stirpe, 1972; McCord 1985). As the ATP is depleted and hypoxanthine
accumulates, upon reperfusion, hypoxanthine is converted to xanthine with
generation of oxygen free radicals such as superoxide, hydroxyl radical and
hydrogen peroxide by xanthine oxidase (McCord 1985). Recent studies have

demonstrated that the microvascular reperfusion injury induced by clamping the

portal triad (hepatic artery, portal vein and common bile duct) is triggered by
superoxide radicals derived from the xanthine oxidase system (Muller et al.,
1996). Liberation of these substances leads to denaturation of proteins and
polyunsaturated fatty acids. When severe, enzyme dysfunction, lipid
peroxidation and membrane disruption can occur.

Superoxide, hydrogen peroxide and the hydroxyl radical are the major
biologically active oxygen species (Reilly 1991). During physiologic
homeostasis, these oxygen radicals are generated in low concentrations and
protective biochemical mechanisms are in place to neutralize them. After a
period of ischemia, followed by reperfusion, the rate of synthesis of these ROS
far exceeds the capacity to convert them to inert molecules. Superoxide is
thought to stimulate changes in the vascular endothelium that produce conditions
that are conducive for the generation of the inﬂammatory response.

In contrast, nitric oxide (NO) production, which is stimulated by IR injury, is
protective. Nitric oxide attenuates sinusoidal perfusion failure, improves liver
oxygenation, and may scavenge oxygen radicals (Lichtman and Lemasters,
1999). Nitric oxide synthesis can be rapid in endothelial cells in response to
shear stress (Kuchan et al., 1994) and during hepatic hypoperfusion (Liu et al.,
1998). Inhibition of NO synthase increases hepatocellular death and increases
liver enzyme release after hepatic IR (Wang et al., 1995; Pannen et al., 1998).
Nitric oxide scavenges ROS by reacting with superoxide to form peroxynitrite.
With increased IR injury, NO synthase inhibition not only increases net

superoxide production, but also increase the gene expression of P-selectin and

lCAM-1, thereby promoting the adherence of neutrophils and the late phase of
hepatic IR injury (Kobayashi et al., 1995; Liu et al., 1998).
Hepatic lschemia/Reperfusion Injury: Late Phase

Neutrophil inﬁltration mediates the late phase of hepatic IR injury. Once
neutrophils reach the site of injury, they amplify and perpetuate the injury by
releasing some of the same proinﬂammatory mediators that attracted them in the
ﬁrst place (Lichtman and Lemasters, 1999). In a study utilizing anti-neutrophil
antibodies, hepatocyte necrosis decreased from 80% to 28% after 24 hours of IR
injury (Jaeschke et al., 1990). Furthermore, direct infusion of neutrophils into
isolated livers after warm IR increases hepatocellular damage, oxygen uptake
and formation of the superoxide radical (Bilzer and Lauterburg, 1994).

The mechanism of hepatocellular injury by neutrophils includes three
steps. The sequence of events begins with activation of neutrophils and their
sequestration in the hepatic vasculature. This process occurs predominantly in
the sinusoids and appears more a passive mechanical trapping than an adhesion
molecule mediated event (Jaeschke et al., 1997; Jaeschke and Smith, 1997).
Next, neutrophils undergo transendothelial migration if the sinusoidal lining is
intact. This process, which is dependent on [32 integrins/ICAM-1 and B1
integrinsNCAM-1, takes place more in the sinusoids and appears to be less
important in postsinusoidal venules (Chosay et al., 1997). The ﬁnal step is the
adherence to the parenchymal cell, which can be dependent on macrophage-1
antigen (Mac-1)/unknown ligand and lymphocyte function-associated antigen-1

(LFA-1)/ICAM-1 (Jaeschke and Smith, 1997). Subsequent hepatocellular

10

necrosis is dependent upon the release of the proteases cathepsin G and
elastase from the adherent neutrophils.
lntercellular Adhesion Molecules

In the late phase of hepatic IR injury, neutrophils ﬁrst marginate to the
sinusoidal wall and subsequently migrate across the endothelium into the space
of Disse (Lichtman and Lemasters, 1999). If the endothelial cell damage is
severe, neutrophils may have direct access to parenchymal cells without
undergoing transmigration. However, when the endothelial cell barrier is intact,
transmigration requires intercellular adhesion molecules. The activation and
recruitment of neutrophils into the liver is the critical step in the pathogenesis of
IR injury.

lntercellular adhesion molecules are cell surface glycoproteins that are not
only involved in cell-cell interactions, but also cell-matrix interactions as well.
These molecules are crucial for leukocyte adhesion to the endothelium,
transmigration, binding to target cells and cytotoxicity (Jaeschke et al.,1997). On
the basis of structure and sequence homology, there are three main families of
adhesion molecules that participate in inﬂammatory processes: integrins, the
immunoglobulin gene superfamily, and selectins.

The role of adhesion molecules has been most extensively studied in
animal models of neutrophil-induced organ injury (Jaeschke et al., 1997).
Investigations utilizing in vitro systems and intravital microscopy have identiﬁed
several discrete steps involving the different adhesion molecules (Granger 1994;

Kishimoto 1994). Since these events take place in the postcapillary venules, the

11

rapidly ﬂowing neutrophils must be slowed down. This is accomplished by the
expression of selectins. Following a rapid upregulation of P-selectin by
mobilization of Weibel-Palade bodies in endothelial cells there is transcriptional
activation of both P-selectin and E-selectin (McEver et al., 1995). The interaction
between selectins expressed on endothelial cells with their ligands on neutrophils
results in tethering and rolling of neutrophils along the vessel wall. During this
hindered movement in the vicinity of an inﬂammatory focus, the neutrophils are
exposed to a multitude of chemotactic factors, including cytokines, chemokines,
complement and platelet activating factor. These inﬂammatory mediators induce
upregulation of Mac-1 and increase the afﬁnity of LFA-1 (CD11a/CD18) on
neutrophils (Jaeschke and Smith, 1997). In addition, it has been shown that
binding of neutrophil L-selectin to its ligand induced up-regulation of Mac-1
adhesion molecule expression on the surface of neutrophils (Crockett-Torabi et
al., 1995). Further, cytokines can induce the expression of ICAM-1 on
endothelial cells through transcription. As a result, these events lead to ﬁrm
adhesion of neutrophils to the vessel wall mainly through interactions between [3;
integrins and lCAM-1 (Granger and Kubes, 1994; Smith 1992). The next step is
the migration of neutrophils through the endothelial cell barrier toward the
inﬂammatory focus, which is facilitated by gradients of chemotactic factors.
Transendothelial migration not only requires 82 integrin-lCAM-1 interaction, it also
requires platelet-endothelial cell adhesion molecule-1 (PECAM-1), which is

expressed on both neutrophils and endothelial cells (Wakelin et al., 1996). While

12

this mechanism applies to several organs, including heart, intestine, skeletal
muscle and skin, there are a few signiﬁcant differences in the liver.
Regulation of Hepatic Adhesion Molecule Expression

The integrin family of adhesion molecules consists of membrane
glycoproteins that are made up of a8 heterodimers (Ruoslahti 1991). Two
integrin receptor families that are important for inﬂammatory reactions deﬁne the
very late antigens (VLA) (B1) and the leukocyte integrins ((32). The very late
antigens and the leukocyte integrins are found on the surface of many cells
including Kupffer cells, pit cells, monocytes, T lymphocytes and neutrophils
(Bioulac—Sage et al., 1996). Quantitatively, the 82 integrin LFA-1(CD11a/CD18)
is highly expressed on all leukocytes (Jaeschke et al., 1997). However, Mac-1
(CD11b/CD18) is present primarily on neutrophils, Kupffer cells, and monocytes.
It is present to a lesser degree on pit cells and is not expressed on T cells
(Jaeschke et al., 1997; Bioulac—Sage et al., 1996). Very late antigen-4 (VLA-4)
(CD49d/C029), a B1 integrin, is expressed mainly on T cells, pit cells and
monocytes, and less on neutrophils and Kupffer cells (Bioulac-Sage et al., 1996;
Essani et al., 1997).

The expression of integrins is regulated at multiple levels. First, synthesis
of LFA—1 and Mac-1 takes place during hematopoietic maturation (Miller et al.,
1986). Second, a further increase of cell surface receptors can occur during cell
activation (Strassman et al., 1985). Finally, cell activation can lead to

conformational changes in the adhesion molecule, resulting in exposure of ligand

13

binding sites. An increase in afﬁnity after stimulation has been demonstrated for
both LFA—1 (Keizer et al., 1988) and VLA-4 (Shimizu et al., 1990).

The activation of integrins, particularly Mac-1, was observed in
pathophysiological situations relevant to hepatic injury in vivo. Macrophage-1
antigen expression on circulating neutrophils was demonstrated in several
models of inﬂammation including hepatic ischemia (Jaeschke et al., 1993),
endotoxemia (Essani et al., 1995; Witthaut et al., 1994) and sepsis (Zhang et al.,
1994). The inﬂammatory mediators that upregulate Mac-1 in these conditions
include activated compliment factors, TNFa and platelet activating factor (PAF)
(Essani et al., 1995; Witthaut et al., 1994; Zimmerman and McIntyre, 1988).

The immunoglobulin gene superfamily includes ICAM-1, lCAM-2, lCAM-3,
vascular endothelial adhesion molecule-1 (VCAM-1) and PECAM-1. In general,
ICAM-2, lCAM-3 and PECAM-I are constitutively expressed on vascular lining
cells, while ICAM-1 and VCAM-1 are both inducible (Jaeschke et al., 1997). In
normal liver, there is low constitutive expression of lCAM-1 on the entire hepatic
endothelium and Kupffer cells (Essani et al., 1995; Farhood et al., 1995;
Jaeschke et al., 1996; Mochida et al., 1996; Ohira et al., 1995; Scoazec and
Feldmann, 1994; Steinhoff and Brandt, 1996). However lCAM-1 is not detected
on hepatocytes (Essani et al., 1995; Farhood et al., 1995; Ohlinger et al., 1993;
Scoazec et al., 1994; Steinhoff and Brandt, 1996) or stellate cells (Hellerbrand et
al., 1996). During inﬂammation, ICAM-1 is strongly upregulated on virtually all
liver cells, including all endothelial cells, Kupffer cells, stellate cells and

hepatocytes (Essani et al., 1995; Farhood et al., 1995; Jaeschke et al., 1996;

14

Mochida et al., 1996; Ohira et al., 1995; Scoazec et al., 1994; Steinhoff 1996;
Hellerbrand 1996; Volpes 1990). Studies with isolated hepatocytes (Kvale and
Brandzaeg, 1993; Mickelson et al., 1995; Morita et al., 1994) and endothelial
cells (Ohira et al., 1994) have demonstrated that TNFa, IL-1 and interferon-y can
induce ICAM-1 expression.

There is low VCAM-1 expression on endothelial cells of arteries and veins,
as well as on bile duct epithelial cells in control livers. But sinusoidal endothelial
cells are very weakly stained (Essani et al., 1997). After administration of
endotoxin, TNFa, or IL-1 in vivo, there is increased staining of VCAM-1 on all
endothelial cells and Kupffer cells (Essani et al., 1997; Van Oosten et al., 1995).
There has been no constitutive or inducible expression of VCAM-1 identiﬁed on
hepatocytes (Essani et al., 1997; Van Oosten et al., 1995).

Platelet-endothelial cell adhesion molecule-1, which is constitutively
expressed on the endothelial cells of large vessels in both human and rodent
livers, cannot be Induced by endotoxin or cytokines (Jaeschke 1997; Scoazec et
al., 1991). It has been demonstrated to be important for leukocyte
transendothelial migration in extrahepatic tissue (Jaeschke and Smith, 1997;
Wakelin et al., 1996).

The selectin family consists of three closely related cell surface molecules
with differential expression by leukocytes (L-selectin, CD62L), platelets (P-
selectin, CD62P), and vascular endothelium (E-selectin, CDS2E). Each of these

molecules contains a calcium-dependent Iectin domain, an epidermal growth

15

factor-like domain and several complement binding repeats as the extracellular
portion of the molecule (McEver et al., 1995; Tedder et al., 1995).

L-selectin is constitutively expressed on neutrophils and is shed from the
cell surface during the exposure of the neutrophil to chemotactic factors that
upregulate Mac-1 (Kishimoto et al., 1989). P-selectin is stored in a-granules of
resting platelets and Weibel-Palade bodies in endothelial cells (Hsu-Lin et al.,
1984; McEver et al., 1989). Within minutes after activation by inﬂammatory and
thrombogenic mediators, P-selectin is mobilized to the cell surface. These 1
mediators include histamine, complement, ROS, cytokines and thrombin (Tedder
et al., 1995). Expression of P-selectin on the cell surface is short-lived.
However, in vivo studies of P-selectin function suggest that it may also be
important at later time points as a cytokine-induced adhesion molecule (Tedder
et al., 1995). Studies have shown that P-selectin is not constitutively expressed
in liver sinusoids and cannot be rapidly upregulated (Essani et al., 1998). The P-
selectin gene can be activated in liver endothelial cells in response to endotoxin
and cytokines (TNFa), but its expression is limited to the endothelium of large
vessels and is not detected in sinusoids (Essani et al., 1995; Tedder et al., 1995).
E-selectin is not found on normal human livers (Adams et al., 1994; Keelan et al.,
1994; Redl et al., 1991). However, during septic shock, endotoxemia or
administration of lL-1, there is expression of E-selectin on large-vessel
endothelial cells and to a lesser degree on sinusoidal lining cells (Keelan et al.,
1994; Redl et al., 1991). E-selectin protein production is also rapidly induced by

TNFa, interferondy and substance P (Bevilacqua et al., 1987).

16

A common feature of the inducible adhesion molecules described (ICAM-
1, VCAM-1, P-selectin and E-selectin) is that each can be transcriptionally
activated by TNFa and lL-1 (Essani et al., 1995, Essani et al., 1997, Essani et
al., 1998). Both cytokines are strong inducers of the transcription factor NFKB
and all inducible adhesion molecules have at least one NFKB binding site in their
promoter region (Collin-et al., 1995). However, only ICAM-1 is upregulated in all
cell types, with VCAM-1, P-selectin and E-selectin only induced in endothelial
cells. This suggests that NFKB activation is necessary but not sufﬁcient to
enhance adhesion molecule expression (Jaeschke 1997). Thus a combination of
several transcription factors may determine which adhesion molecule is
upregulated in each liver cell type.
Adhesion Molecules and Neutrophil-Induced Liver Damage

Recent reports have demonstrated role for P-selectin in hepatic IR injury
utilizing monoclonal antibody (mAb) blockade. Garcia-Criado et al. (1995)
administered a mAb to P-selectin before and after reperfusion in a rat model of
partial hepatic ischemia and showed reduced hepatic damage, decreased
myeloperoxidase activity and improved survival. Another group of investigators
that employed an anti-P-selectin mAb not only conﬁrmed attenuated
hepatocellular damage in a model of IR, but also found decreased neutrophil
accumulation (Singh et al., 1998). In a study using soluble P-selectin
glycoprotein ligand-1(PSGL-1), a ligand for P-selectin, given at the time of
hepatic inﬂow occlusion in a warm ischemia model, there was reduced aspartate

aminotransferase (AST), hepatic damage and neutrophil inﬁltration

17

(Dulkanchainun et al., 1998). Furthermore, there was improved survival when
the soluble PSGL-1 ligand was administered before cold ischemia and at the
time of reperfusion in a transplanted liver.

One investigation, which utilized P-selectin deﬁcient mice in a model of
warm partial hepatic ischemia, demonstrated an important role for P-selectin in
neutrophil and platelet sequestration (Yadav et al., 1999). Following 90 minutes
of ischemia, there was a signiﬁcant decrease in serum transaminase levels,
hepatocellular necrosis, as well as neutrophil inﬁltration and adhesion (Yadav et
al., 1999). In a model of total hepatic ischemia, there was improved survival in
mice subjected to both 75 and 90 minutes of ischemia (Yadav et al., 1999). In
addition, platelet sequestration, as determined by in situ immunohistochemical
staining, was markedly reduced in the P-selectin deﬁcient mice (Yadav et al.,
1999)

In other studies using P-selectin knockout mice in a model of partial
hepatic ischemia, intravital microscopy was employed to investigate leukocyte
recruitment in the postischemic hepatic vasculature. An increase in rolling
leukocytes was observed in wild-type mice subjected to 20 minutes of ischemia
and 5 hours of reperfusion (Zibari et al., 1998). This phenomenon was not
observed in control mice, P-selectin deﬁcient mice, or wild-type mice treated with
a mAb to P-selectin (Zibari et al., 1998). Furthermore, when this same group
looked at the number of rolling, saltating and adherent leukocytes in terminal

hepatic venules after IR, there were signiﬁcant increases in wild-type mice,

18

whereas in P-selectin deﬁcient mice these responses were absent (Sawaya et
al., 1999).

Some studies have concluded that P-selectin, in contrast to many other
organs, is not required for sinusoidal neutrophil sequestration, transendothelial
migration, and subsequent neutrophil-induced liver parenchymal injury. Since
there are no WeibeI-Palade bodies in sinusoidal endothelial cells of normal liver
(Wisse 1972; Irving et al., 1984), P-selectin expression in the liver must be under
transcriptional control (Essani et al., 1998). In a model of endotoxemia, P-
selectin mRNA formation was demonstrated in Kupffer cells as well as
endothelial cells (Essani et al., 1995). In a model of galactosamine and
Salmonella abortus equi endotoxemia, immunohistochemical analysis indicated
that P-selectin was expressed on endothelial cells of the periportal and central
vein areas, with no P-selectin detected on sinusoidal endothelial cells (Essani et
al., 1998). Furthermore, administration of an anti-P-selectin mAb neither reduced
the number of accumulating neutrophils in the sinusoids, nor attenuated liver
injury. I

A study that focused on the role of L-selectin and lCAM-1 in hepatic IR
injury, utilized mice that were deﬁcient in L-selectin, ICAM-1, or both. A model of
left lateral lobe IR injury was employed, with ischemia times of 30, 60 and 90
minutes, followed by reperfusion times of 15 minutes, 1 hour, 6 hours and 24
hours. In mice that were deﬁcient in L-selectin alone, there was a wide range of
protective effects on IR injury observed, including decreases in neutrophil

inﬁltration, prevention of microcirculatory failure, lower transaminase levels

19

(Yadav et al., 1998). There was also improved overall survival in a model of total
hepatic ischemia (Yadav et al., 1998). In mice deﬁcient in lCAM-1, the protective
effects were limited to a decrease in both neutrophil inﬁltration and transaminase
levels only at 6 hours of reperfusion (Yadav et al., 1998). The L-selectin/lCAM-1
double mutant mice demonstrated a reduction in injury with a pattern that was
very similar to the L-selectin deﬁcient mice. However, unlike either the L-selectin
or lCAM-1 deﬁcient mice, there was a statistically signiﬁcant reduction in
transaminase levels after 24 hours of reperfusion, suggesting a possible additive
effect (Yadav et al., 1998).
Adhesion Molecule Deﬁcient Mice in Models of IshemialReperfusion

Recent developments in molecular genetic analyses of mice have made it
possible to generate null mutations in any gene of interest (Hynes and Wagner,
1996). Much useful information has already been obtained from such “knockout”
mice. Transgenic mice, to which genes have been added, have been available
for longer and provide another avenue for manipulating the genome of mice.
Moreover, it is now possible to generate subtle mutations and tissue-speciﬁc or
regulatable expression or ablation of speciﬁc genes (Rossant and Naggy, 1995;
Marth 1996). Mice can be interbred to combine mutations in multiple genes,
providing animal models for multigenic defects.

Even though the use of blocking agents is particularly applicable to
designing strategies to prevent IR injury in a speciﬁc organ with potential clinical
application, they provide only limited insight into the mechanism of injury.

Moreover, they do not convincingly establish the involvement of a speciﬁc

20

adhesion molecule. Other ligands may interact with other adhesion molecules or
receptors resulting in nonspeciﬁc stimulatory or inhibitory effects. In particular,
for a substance such as soluble PSGL —1, various studies have shown
concomitant blockage of L-selectin (Spertini et al., 1996; Tu et al., 1996), as well
as E-selectin (Asa et al., 1995).

One of primary advantages of knockout technology over immunological
manipulation is the ability to speciﬁcally and completely ablate the gene product.
This eliminates the potential inherent ability of antibodies to trigger secondary
cellular responses or nonspeciﬁc immunological effects. Antibodies against
adhesion molecules have been shown to mimic the effects of the natural ligands
of the surface target molecule (Schachner 1993). For example, antibodies to
neural cell adhesion molecule activate G-protein—coupled phospholipase C
signaling pathways, increase cytosolic free calcium concentration, decrease
intracellular pH, and activate protein kinases (Schuch et al., 1989). Antibodies to
adhesion molecules have been shown to have multiple effects on signaling and
genetic responses in cells (Schachner 1993). In cells treated with antibodies to
B1 integrins, for example, there is increased tyrosine phosphorylation of a
number of proteins (Kornberg et al., 1991). It is also possible for an antibody to
induce a different set of signals and responses than the actual ligand.
Furthermore, since antibodies vary in their speciﬁcity, blocking a particular
epitope does not necessarily neutralize the functional activity of the protein to
which the antibody is directed. In addition, the local availability of the blocking

mAbs may be compromised, particularly in the presence of extensive occlusion

21

of the vasculature. Targeted gene inactivation technology thus affords the
opportunity to study the role of a speciﬁc molecule in a pure system.

Knockout technology is only in current routine use in mice. The mouse
has a long history of use as a key biological tool for inﬂammation research. The
advantages include its well-studied genetics, short reproductive cycle and well-
characterized models. Disadvantages relate to the small size of mice for some
sample preparation and variability in susceptibility to inﬂammatory stimuli due to
genetic strain differences. Nonetheless, signiﬁcant progress in the
understanding of the mechanisms of inﬂammation and the immune response has
been gained in the use of knockout mice.

Neurologic lschemia/Reperfusion Injury

There is accumulating evidence that cerebral ischemia elicits an
inﬂammatory response that is augmented by reperfusion. Leukocyte inﬁltration
has been well documented after cerebral IR and is known to mediate local tissue
damage as well as alterations in microvascular perfusion (Frenette and Wagner,
1996; Garcia et al., 1994; Hallenbeck et al., 1985; Kochanek and Hallenbeck,
1992). In a murine model of reperfused stroke, it was demonstrated that
neutrophil depletion before stroke reduced cerebral infarct volume by three-fold
and improved functional outcome (Connolly et al., 1996).

To investigate the role of P-selectin in stroke, Connolly, et al. utilized a
murine model of focal cerebral IR via middle cerebral artery occlusion in
homozygous P-selectin null mice and a strategy of administering a functionally

blocking P-selectin antibody. Expression of P-selectin in the postischemic

22

cerebral cortex was localized to the ipsilateral cerebral microvascular endothelial
cells by immunohistochemistry and by speciﬁc accumulation of radiolabeled anti-
murine P-selectin lgG. Furthermore, neutrophil accumulation in the ischemic
cerebral cortex of mice expressing the P-selectin gene was signiﬁcantly greater
than that of the P-selectin null mice. These mice also demonstrated smaller
infarct volumes and improved overall survival compared to the wild-type mice
(Connolly et al., 1997). In addition, functional mAb blockade of P-selectin in wild-
type mice led to improved early reﬂow and stroke outcome compared with control
mice. Cerebral infarction volumes were reduced even when the blocking
antibody was administered after the occlusion of the middle cerebral artery
(Connolly et al., 1997).

Another investigation evaluated the role of P-selectin and E-selectin in a
model of transient focal cerebral IR utilizing a double knockout mouse that was
deﬁcient in both selectins. After 3 hours of ischemia via MCAO and 21 hours of
reperfusion, there was no difference in infarct volume (Soriano et al., 1999).

Other studies have implicated an important role for lCAM-1 in neutrophil-
mediated cerebral IR injury. lCAM-1 is upregulated on microvascular
endothelium and glial cells during inﬂammatory processes in the brain (Fabry et
al., 1992; Frohman et al., 1995). Speciﬁc blockade of lCAM-1 by a mAb has
been shown to attenuate cerebral damage after transient focal ischemia (Zhang
et al., 1994). Two studies utilizing an lCAM-1 deﬁcient mouse in a model of
transient focal cerebral IR both demonstrated a reduction in infarct volume

compared to wild-type controls (Connolly et al., 1996; Soriano et al., 1996). In

23

the various models of cerebral IR injury employed in these investigations, there
was a 3.7 to 7.8-fold reduction in infarct volume in the ICAM-1 null mice. In
addition, lCAM-1 null mice demonstrated a 35% increase in survival and reduced
neurologic deﬁcit compared with wild-type controls (Connolly et al., 1996). An
important role for ICAM-1 in the genesis of cerebral no-reﬂow was suggested by
the ﬁnding that cerebral blood ﬂow to the infarcted hemisphere was 3.1-fold
greater in lCAM-1 null mice compared to wild-type controls (Connolly et al.,
1996)

The role of Mac-1 in neutrophil inﬁltration after transient focal IR has been
investigated as well. In studies using a mAb to Mac-1 there was a reduced
Infarct volume after IR (Chen et al., 1994; Springer 1995). However,
administration of this antibody was not without complication, as a partial
depletion of peripheral white blood cells was noted. Investigations that utilized a
mAb to the CD18 subunit have yielded conﬂicting results. In a feline model, the
antibody had no effect on infarct volume or cerebral blood ﬂow (Takeshima et al.,
1992). Yet in a primate model, there was improved microvascular patency after
IR (Mori et al., 1992). In a study of cerebral using Mac-1 deﬁcient mice, there
was a signiﬁcant decrease in infarction volume by 26%. Furthermore, there was
a reduction in extravasated neutrophils in the infarcted brains of the mutant mice,
though this ﬁnding was not statistically signiﬁcant (Soriano et al., 1999).

Myocardial lschemia/Reperfusion Injury
Myocardial IR injury is considered a potent stimulus for tissue destruction

and possible cardiac failure (Braunwald and Kloner, 1985; Entman et al., 1991).

24

A large extent of this injury is believed to result from intensiﬁed neutrophil-
endothelial cell afﬁnity via enhanced adhesion molecule expression subsequent
to myocardial IR. Clinical investigations have provided some preliminary
evidence that neutrophils and endothelial cell adhesion molecules are activated
in the coronary circulation under conditions of unstable angina (Buerke et al.,
1994; Lefer AM et al., 1994), coronary vasospasm (Eppihimer et al., 1996; Gill et
al., 1996) and acute myocardial infarction (Lefer DJ et al., 1996) in humans.
Studies have suggested a pivotal role for P-selectin in myocardial IR
injury. It has been demonstrated that administration of monoclonal antibodies
(Weyrich et al., 1993) or carbohydrate ligands (Lefer DJ et al., 1994) directed
against P-selectin markedly attenuates myocardial reperfusion injury in animal
models of myocardial IR. Palazzo et al. (1998) utilized P-selectin knockout mice
to evaluate the role of P-selectin in myocardial IR injury. After 30 minutes of
ischemia followed by 120 minutes of reperfusion, there was signiﬁcant diminution
of myocardial infarct size and neutrophil accumulation in the P-selectin deﬁcient
mouse heart compared to the wild-type mouse hearts. However, with prolonged
coronary artery occlusion, (Le. 60 minutes) the infarct size was increased, but
similar between P-selectin-deﬁcient and wild-type mice. Yet there was no
difference in PMN inﬁltration between 30 minutes and 60 minutes of myocardial
ischemia. Therefore P-selectin deﬁciency may not confer cardioprotection during
extended periods of coronary artery occlusion, which may be attributed to

neutrophil-independent myocardial necrosis.

25

There Is a large amount of experimental evidence that suggests CD18
interactions with lCAM-1 play a signiﬁcant role in neutrophil-mediated myocardial
IR injury. The involvement of lCAM-1 in myocardial IR injury has been previously
demonstrated (Kukielka et al., 1993; Youker et al., 1994). In addition, it has also
been shown that neutrophil CD18 participates in the ﬁrm adhesion of activated
neutrophils to cardiac myocytes through interaction its with lCAM-1, leading to
myocyte injury and necrosis (Entman et al., 1992; Smith et al., 1989).
Monoclonal antibodies against the neutrophil [lg-integrin complex CD11/CD18
have been demonstrated to have cardioprotective effects (Aral et al., 1996;
Aversano et al., 1995; Lefer DJ et al., 1993; Ma et al., 1991; Simpson et al.,
1988). Similarly, monoclonal antibodies against lCAM-1, the endothelial ligand
for CD11/CD18, have been shown to decrease the injury produced by myocardial
IR (Hartman et al., 1995; Lefer DJ et al., 1996; Ma et al., 1992; Yamazaki et al.,
1993). However, other studies have reported that anti-CD18 monoclonal
antibodies fail to reduce myocardial IR injury.

Palazzo et al. (1998) examined the role of CD18 and lCAM-1 individually
in a model of murine myocardial IR utilizing knockout mice. Mice were subjected
to 30 minutes of myocardial ischemia and 120 minutes of reperfusion to
determine the extent of myocardial cell necrosis and neutrophil inﬁltration.
Myocardial infarction, as calculated in % of the area at risk, was 45.1 +/- 5.9 in
wild-type mouse hearts. The extent of myocardial infarction was signiﬁcantly
attenuated in the CD-18-deﬁcient mice (i.e. 19.3 +/- 5.1%), as well as in the

lCAM-1-deﬁcient mice (i.e. 17.9 +/- 3.2%). Similarly, neutrophil inﬁltration into

26

the IR myocardium was reduced by 54% in the CD18-deﬁcient mice and by 32%
in ICAM-1-deﬁcient mice compared to wild-type hearts. Based on these data,
there is strong evidence that both of these adhesion molecules play an important
role in myocardial cell injury in IR.
Lung lschemia/Reperfusion Injury

Several studies have demonstrated P-selectin-mediated pulmonary PMN
sequestration in models of remote tissue IlR injury, including intestine and
skeletal muscle (Carden et al., 1993; Seekamp et al., 1994). However, one study
investigated the role of P-selectin in pulmonary injury in models of hypothermic
preservation with transplantation and direct ischemic insult. To determine
whether P-selectin played a role in mediating early primary graft failure, left lungs
harvested from male Lewis rats were preserved for 6 hours at 4°C and
transplanted orthotopically into isogenic recipients. Recipients immunodepleted
of neutrophils prior to transplantation demonstrated improved graft function and
overall survival. Administration of a blocking P—selectin antibody 10 minutes prior
to reperfusion diminished graft PMN inﬁltration and improved graft function as
evidenced by reduced pulmonary vascular resistance and increased pulmonary
arterial ﬂow. Recipient survival was improved as well.

To evaluate the role of P-selectin in normothermic pulmonary ischemia,
mice were subjected to temporary left pulmonary artery ligation (Le. 30 or 60
minutes) and 30 minutes of reperfusion. After functional removal of the
nonischemic right lung, P-selectin-deﬁcient mice demonstrated reduced PMN

inﬁltration, improved arterial oxygenation and improved survival compared to

27

controls. These studies indicate a central role of P-selectin in PMN recruitment
and tissue injury in the setting of lung transplantation after hypothermic
preservation and after normothermic ischemia. (Naka et al., 1997)

Renal lschemia/Reperfusion Injury

Studies in the rat utilizing mAbs have suggested a role for lCAM-1 in the
pathogenesis of acute tubular necrosis. However, anti-lCAM-1 mAb may not
block all lCAM-1-dependent interactions since lCAM-1 has multiple sites for
integrin binding (Springer 1990). Furthermore, antibodies vary in their speciﬁcity
and blocking a particular epitope does not necessarily neutralize the functional
activity of the protein to which the antibody is directed. Since not all of the
functions of ICAM—1 are known, it is not possible to determine in vivo or in vitro
the extent to which the antibody is neutralizing.

Kelly et al. (1996) studied the role of lCAM-1 in ischemic renal injury by
determining the histological and functional consequences of bilateral IR injury in
mice genetically deﬁcient in lCAM-1 expression. Serum creatinine was
signiﬁcantly increased over baseline after bilateral renal ischemia and
reperfusion at 24, 48 and 72 hours in control mice. lntercellular adhesion
molecule-1-deﬁcient mice were protected from renal IR injury. After 72 hours of
reperfusion, all of the lCAM-1-deﬁcent mice survived, whereas 50% of the control
wild-type animals died. Histologic analysis of the kidneys of wild-type mice
revealed signiﬁcantly more tubular epithelial necrosis in the outer medulla than
lCAM-1 deﬁcient mice. Control wild-type mice had signiﬁcantly more neutrophils

in the outer medulla, as well as greater myeloperoxidase activity than lCAM-1-

28

deﬁcient mice did. These data suggest an important role for ICAM-1 and
leukocyte-endothelial adhesion in the pathophysiology of ischemic renal failure.
P-selectinIICAM-1 Double Mutant Mice in Models of Inﬂammation

Bullard et al ﬁrst described P-selectin/ICAM-1 double mutant mice in 1995.
P-selectin mutant mice were generated by gene targeting using a replacement
vector designed to delete exons 3-5. Deletion of a 4.5-kb region containing these
exons was conﬁrmed by Southern blotting and reverse transcriptase polymerase
chain reaction. Mutant animals demonstrated a loss of expression on both
endothelium and platelets. P-selectin mutant mice were bred to mice containing
a mutation in the lCAM-1 gene within exon 5 (Sligh et al., 1993) to generate
double homozygotes.

Examination of circulating leukocyte counts demonstrated a signiﬁcant
increase in the number of neutrophils in P-selectinllCAM-l double mutants when
compared to mice of similar genetic background. The circulating lymphocyte and
monocyte counts were increased as well.

The ﬁrst study with these mice investigated the roles of P-selectin and
lCAM-1 in the acute inﬂammatory response induced by S. pneumoniae within the
peritoneum and lung. In a model of S. pneumoniae peritonitis, there was a
complete absence of neutrophil emigration in the peritoneum in the P-
selectin/lCAM-1double mutant mice, compared to a 60-70% reduction with either
mutation alone (Bullard et al., 1995). The data indicate that the roles of P-
selectin and ICAM-1 are unlikely to be completely independent in this model. In

contrast, the combined absence of these molecules had no effect on neutrophil

29

emigration into the parenchyma of the lung in response to the same stimulus.
These data suggest that neutrophil emigration through the pulmonary
microvasculature can occur through adhesion pathways that do not require P-
selectin or lCAM-1.

Another investigation examined the role of P-selectin and lCAM-1 in
traumatic brain injury utilizing P-selectin/lCAM-1 double mutant mice.
Experimental traumatic brain injury elicits an acute local inﬂammatory response,
including up-regulation of adhesion molecules and accumulation of neutrophils,
in injured brain (Carlos et al., 1997; Whalen et al., 1997). In a model of traumatic
brain injury there was no difference in neutrophil accumulation between P-
selectin/lCAM-1 double knockout mice and wild-type mice. However, brain
edema was signiﬁcantly decreased in the deﬁcient mice. In comparing
histopathology and tests of memory and motor function, no signiﬁcant differences
between the two groups were identiﬁed (Whalen et al., 2000). These data
suggest a role for adhesion molecules in the pathogenesis of brain edema

independent of leukocyte accumulation (Whalen et al., 2000).

30

MATERIALS AND METHODS

Animals

Gene-targeted mice deﬁcient in P-selectin and lCAM-1 (P/l null), C57BL/6-
lcam1WIBaySelpm‘Bay, were purchased from the Jackson Laboratory (Bar Harbor,
ME). These mice demonstrate an increased number of neutrophils in the blood
and a complete loss of neutrophil emigration into the peritoneum during
Streptococcus pneumoniae-induced peritonitis (Bullard 1995). Homozygotes are
viable and fertile with to obvious phenotypic abnormalities. Male double-
knockout mice were either bred under the guidance of the University Laboratory
Animal Resources, Michigan State University, or purchased directly from the
Jackson Laboratory. Wild-type mice were C57BL/6 from Charles River
Laboratories, Portage, MI. Mice were maintained on 12-hour light and 12-hour
dark cycles. The animals had access to food and water ad libitum. All
procedures were performed according to the Michigan State University All-
University Committee on Animal Use and Care guidelines.
Model of Partial Hepatic lschemia/Reperfusion Injury

A murine model of lobar hepatic ischemia was utilized (Colletti et al., 1990;
Kawano et al., 1989; Lichtman and Lemasters, 1999, Martinez-Mier et al., 2000).
Mice were anesthetized with 1-2 ml of methoxyﬂurane (Schering-Plough, Union,
NJ) applied to gauze in a face cone, which was fashioned from a 50-ml Corning
polypropylene disposable centrifuge tube (Corning, NY). Once adequate
anesthesia was obtained, as determined by a negative foot pinch reﬂex, an

intraperitoneal injection (15 mg/kg) of pentobarbital sodium (50mg/ml, Abbott

31

Laboratories, North Chicago, IL) was administered. The face cone was applied
as needed to maintain anesthesia. A midline incision was made from the xiphoid
process to the pubis. The small and large bowel were eviscerated and packed in
normal (0.9%) saline moistened gauze (2 in. x 2 in., The Kendall Company,
Mansﬁeld, MA). The liver was exposed completely. The ligamentous attachment
of the left lateral lobe was carefully divided. The median and left lateral lobes
were freed. The portal circulation to both of these lobes was carefully dissected.
The portal vein and hepatic artery supplying the median and left lateral lobes was
then interrupted with the application of an atraumatic vascular clamp (accurate
Surgical and Scientiﬁc Instruments Corporation, Westbury, NY) to the vascular
pedicle. The caudate and right lateral lobes, as well as the papillary and
quadrate processes of the liver were perfused to prevent intestinal congestion.
This results in the induction of ischemia to approximately 70% of the liver
(Kawano et al., 1989; Lichtman and Lemasters, 1999). The small and large
bowel were replaced into the abdominal cavity. One ml of sterile normal saline
was dripped into the abdominal cavity to replace insensible ﬂuid losses and
prevent desiccation. During hepatic ischemia, the abdomen was covered with
plastic wrap (S. C. Johnson & Son, Inc., Racine, WI) to prevent evaporation.
After 90 minutes of partial hepatic ischemia, the clamp was removed and
reperfusion was initiated. The midline Iaparotomy was closed in a single layer
using 5-0 nylon on an FS-2 needle (Ethicon, lnc., Somerville, NJ). Sterile
Lactated Ringers solution (Abbott Laboratories, North Chicago, IL), 0.8 ml, was

administered subcutaneously to compensate for operative blood and ﬂuid losses.

32

Sham mice underwent the same procedure but without vascular occlusion. Mice
were sacriﬁced after 0, 1.5, 3 and 6 hours of reperfusion. The experimental

procedure is summarized in Figure 1.

33

Median Lobe

   

Left Lateral
Lobe

    

Papillary and
Quadrate Processes
Caudate and Right
Lateral Lobes

Figure 1. Model of Partial Hepatic lschemia
The blood supply to the median and left lateral lobes Is interupted with an
atraumatic microvascular clamp. The total ischemia time is 90 minutes, followed

by various reperfusion times.

Determination of Plasma Alanine Aminotransferase Levels

Plasma levels of ALT were utilized as an established marker of hepatic I/R
injury. Blood samples were obtained from the right ventricle via a left anterior
thoracotomy at the time of sacriﬁce. The blood was collected in a sterile
heparinized (50pl, 100 USP Units/ml, Abbott Laboratories, North Chicago, IL) 3
cc syringe (Becton Dickinson & 00., Franklin Lakes, NJ) with a 25G needle
(Becton Dickinson & 00., Franklin Lakes, NJ). The blood was centrifuged
(Eppendorf Model 5415C, Brinkmann Instruments, Inc., Westbury, NY) at 14,000
rpm in 18-20°C for 15 minutes. The plasma was then transferred to two sterile
1.5 ml eppendorf microcentrifuge tubes (Brinkmann Instruments, Inc., Westbury,
NY) using a sterile transfer pipette (Sarstedt, Inc., Newton, NC). The plasma
was then stored at —70°C until further use.

Measurement of plasma ALT was performed using a diagnostic kit from
Sigma Chemical Company (St. Louis, MO). This assay is based on colorimetric
measurement of transaminase in plasma by formation of 2, 4-
dinitrophenylhydrazones of the keto-acids produced by the enzymes (Reitman
and Frankel, 1957). Brieﬂy, 1.0 ml of Alanine-a-KG Substrate (Sigma Chemical
Company, St. Louis, MO) was pipetted into disposable glass culture tubes (VWR
Scientiﬁc, Chicago, IL). Plasma samples were diluted in 0.9% saline. Next 200
pl of each plasma sample was added to each tube and vortexed (Vortex Genie 2,
Fisher Scientiﬁc, Pittsburgh, PA) for 10 seconds. The samples were then
incubated in a 37°C water bath (Lauda, Model M20, VWR Scientiﬁc, Chicago, IL)

for 30 minutes. Next, 1.0 ml of Sigma Color Reagent was added. The reaction

35

mixture was vortexed and incubated at room temperature for 20 minutes. The
reaction was stopped with 10 ml of 0.40N sodium hydroxide solution (J. T. Baker
Chemical Company, Phillipsburg, NJ). The tubes were then covered with
Paraﬁlm® “M”(American National Can, Menasha, WI) and the contents mixed
using gentle inversion. After 5 minutes, the absorbance was measured at 500
nm using a Spectramax Plus Microplate Reader (Molecular Devices, Sunnyvale,
CA). A standard curve was generated from a plot of the OD. values at 500 nm
versus a known amount of ALT activity per ml (Sigma-Frankel Units/ml) (Sigma
Chemical Company, St. Louis, MO). The plasma concentration of ALT activity In
the experimental samples was then calculated from the standard curve and is
presented as the international unit per liter of plasma (IU/L).
Histological Assessment of Hepatocellular Damage

Portions of the left lateral and caudate lobes were ﬁxed in buffered 10%
formalin and embedded in parafﬁn. Sections were cut to 5 microns, which
underwent routine staining with hematoxylin and eosin. The tissues were
examined by two including individuals, a board certiﬁed veterinary pathologist
(J.H.), who were blinded from the experimental treatment of the individual mice.
lmmunohistochemistry for ICAM-1 and Neutrophlls

lnercellular adhesion molecule-1 expression was analyzed by
immunohistochemistry using an Armenian hamster lgG anti-mouse primary mAb
(3E2) speciﬁc for lCAM-1 (CD54) (01541 D, PharMingen, San Diego, CA).
Biopsies of the ischemic median lobe were placed in intermediate Cryomolds

(Miles lnc., Elkhart, IN) and embedded in optimal cutting temperature (OCT)

36

embedding medium (Sakura Finetek USA, Inc., Torrance, CA). The samples
were then frozen by ﬂoating the specimen molds in 2-methylbutane (Fisher
Scientiﬁc, Pittsburgh, PA) prechilled in liquid nitrogen and then stored at -70°C.
Cryosections were cut at 5 microns, and placed on glass slides (VWR Scientiﬁc,
Chicago, IL) and ﬁxed in acetone. Slides were then placed in a humidiﬁed
chamber. The tissue sections were rinsed with TBS (BioRad, Hercules, CA), pH
7.4, and incubated in Tween-buffered saline(TBS) for 5 minutes. This was
repeated for a total of two washes. To prevent nonspeciﬁc binding, the tissue
sections were incubated in goat serum (Vector Laboratories, Inc., Burlingame,
CA)/hydrogen peroxide (Sigma Chemical Company, St. Louis, M0) for 10
minutes. The tissue sections were then rinsed twice with TBS for 5 minutes per
rinse. Next the tissue sections were incubated with Avidin-D (Vector
Laboratories, Inc., Burlingame, CA) and incubated for 15 minutes, followed by a
wash with TBS. Biotin Blocking Reagent (Vector Laboratories, Inc., Burlingame,
CA) was then applied to the tissue sections and incubated for 15 minutes.
Tissue sections were again washed with TBS for 5 minutes and the excess was
removed. Next 100pl of the anti-ICAM-1 antibody was applied to the tissue and
incubated for 60 minutes. The tissue sections were then rinsed three times with
TBS for a total of 10 minutes and the excess was wiped away. Next 100 pl of the
secondary antibody, biotin-conjugated mouse anti-hamster lgG mAb (12102D,
PharMingen, San Diego, CA) was applied to the tissue and incubated for 30
minutes. A 10-minute TBS rinse was again performed. Vectastain ABC Reagent

(Vector Laboratories, Inc., Burlingame, CA) was applied to the tissue and

37

incubated for 30 minutes, followed by a 10—minute TBS rinse. The 3,3”—
diaminobenzidine (DAB) solution (Vector Laboratories, Inc., Burlingame, CA) was
then applied and incubated for 10 minutes. The tissue cultures were then rinsed
with 0.05M sodium bicarbonate (J. T. Baker Chemical Company, Phillipsburg,
NJ) and incubated for 10 minutes. Next, DAB Enhancing solution was applied for
5-10 seconds per slide and rinsed with dHZO.

The tissues were then stained in Hematoxylin (Gill’s Formula, Vector
Laboratories, I nc., Burlingame, CA) for 5 minutes. The tissue sections were then
rinsed with dH20 until the efﬂuent was clear. Next the tissue sections were
dipped ten times in 2% acetic acid (EM Science, Gibbstown, NJ), followed by
rinsing with dH20. The tissue sections were then placed in a bluing solution (1.5
ml NH4OH (EM Science, Gibbstown, NJ) and 98.5 ml 70% EtOH) for one minute.
Tissue sections were then dipped ten times in dH20 and allowed to air dry.
Finally a coverslip was applied with Dako Mounting Media (Dako Corporation,
Carpinteria, CA)

Immunohistochemical staining for neutrophils was performed on biopsies
of the ischemic lobes embedded in OCT as previously described. The primary
antibody was a rat Inga anti-mouse neutrophil mAb (7/4) (CL8993AP,
Cedarlane, Accurate Chemical & Scientiﬁc Corporation, Westbury, NY); while the
secondary antibody was the biotin-conjugated mouse anti-rat lgG mAb (121020,
PharMingen, San Diego, CA). The procedure was otherwise as described

above.

38

Statistical Analysis

A two-way analysis of variance was utilized to test for differences in ALT
between wild-type and P/l null mice. Analysis was performed using the Number
Cruncher Statistical System (Number Cruncher Statistical Systems, Kaysville,

UT). A p value less than 0.05 was considered statistically signiﬁcant.

39

RESULTS

Veriﬁcation of lCAM-1 Deﬁciency in PII Null Mice

The lCAM-1 expression was assessed in situ by speciﬁc
immunohistochemical staining for lCAM-1 (0054) utilizing a speciﬁc mAb to
mouse lCAM-1. lmmunostaining demonstrated that lCAM-1 was constitutively
expressed in the wild-type control mice as indicated by intense brown staining
along the endothelium of the central vein, sinusoids and portal vasculature
(Figure 2A). In P/l null control mice, ICAM-1 deﬁciency was conﬁrmed by the
lack of staining along the endothelium (Figure ZB). After hepatic IR, lCAM-1
continued to be expressed as demonstrated by the continued intense staining of
the endothelium (Figure 20). Further, there was no evidence of ICAM-1

expression in the PH null mice, even after 6 hours of reperfusion (Figure 20).

40

Figure 2A. Control, wild-type mouse (no ischemia and reperfusion).

Figure 23. Control, P/I null mouse (no ischemia and reperfusion).

Figure 2C. Wild-type mouse after 90 minutes of ischemia and 6 hours of

reperfusion.

Figure ZD. P/I null mouse after 90 minutes of ischemia and 6 hours of

reperfusion.

41

 

42

Demonstration of Hepatocellular Injury by Determination of Alanine
Aminotransferase Levels

Hepatic IR caused signiﬁcant hepatocellular damage in a time-dependent
manner as demonstrated by plasma ALT levels. The plasma ALT levels of both
wild-type and P/l null mice after 90 minutes of ischemia followed by 1.5, 3 and 6
hours of reperfusion were signiﬁcantly elevated compared to the sham operated
mice at the same time points. However, there was no statistically signiﬁcant
difference in ALT levels between the wild-type and PH null mice that underwent
90 minutes of ischemia and 0 hours of reperfusion, compared to the sham group
at 0 hours for both types of mice (Table 1). In addition, there was no signiﬁcant
difference between wild-type and PH null sham operated mice at 0, 1.5, 3 and 6
hours of reperfusion (62 1 10 vs. 49 1 5, 105 1 17 vs. 83 1 13, 78 1 10 vs. 95 1
30, 68 1 15 vs. 56 1 22 IU/L, respectively).

In comparing the wild-type and PH null mice after 1.5, 3 and 6 hour
reperfusion, there was a trend toward decreased injury in PM null mice compared
to the wild-types (1977 1 289 vs. 2090 1 472, 3877 1 807 vs. 4267 1 489, 4233 1
495 vs. 5613 1 576 lU/L, respectively) (Figure 3). However, this was not

statistically signiﬁcant.

43

Table 1. Summary of Plasma ALT Levels in Wild-Type and P/l Null Mice After
Sham Operation and IR. Values are expressed as the mean 1 standard error of
the mean (SEM) with n = 3 for sham groups and n z 5 for the IR groups.

 

 

Reperfusion Time Treatment Wild-Type PII Null
0h Sham 62110 4915
IR 86 1 13 66 1 22
15h Sham 105117 83113
IR 2090 1 472 1977 1 290
3 h Sham 78 110 95 1 30
IR 4267 1 489 3877 1 807
6 h Sham 68 115 56 1 22
IR 5613 1 576 4233 1 495

44

7000 ‘

6000 ‘

5000 ‘

4000 ‘

3000 '1

ALT (lU/L)

2000 ‘

1000 ‘

 

O-

UWild-type
I P/l Null

 

 

 

 

 

 

   

0 1.5 3 6
Reperfusion Time (hrs)

Figure 3. Hepatocellular Injury After 90 Minutes of Hepatic lschemia With

Various Reperfusion Times - Wild-Type vs. P/l Null Mice. Values are expressed

as the mean 1 SEM with n _>_ 5 in each group.

45

Demonstration of Hepatocellular Injury by Histopathology — Hematoxylin
and Eosin Staining

The liver lobule consists of three main regions. The periportal area
includes the portal triad, which is comprised of the portal vein (PV), hepatic artery
and bile duct. The pericentral area surrounds the central vein (CV). The
midzonal region is found between the periportal and pericentral areas.

Histopathologic evidence of hepatic IR injury is classically includes
sinusoidal congestion, cytoplasmic vacuolization, hepatocellular necrosis and
neutrophil inﬁltration (Suzuki et al., 1994). Typically, after ischemia followed by a
long period of reperfusion, there is relative sparing of the periportal region, and
signiﬁcant necrosis in the midzonal and pericentral regions. In this study, after
90 minutes of ischemia followed by 0 hours of reperfusion, there is minimal
evidence of hepatocellular injury (Figure 4A). However, following 1.5 and 3
hours of reperfusion there was moderate hepatocellular necrosis in both the wild-
type and P/I null mice, and marked hepatocellular necrosis after 6 hours of
reperfusion (Figure 4B and 4C). As ﬁgure 4B shows, a greater number of
neutrophils inﬁltrated into the midzonal region of ischemic liver after 6 hours of
reperfusion in wild-type mice. There was no evidence of hepatocellular injury in
the corresponding non-ischemic lobes of the livers of both animal groups that

underwent 90 minutes of ischemia and 1.5, 3, and 6 hours of reperfusion.

46

Figure 4A. Control, wild-type mouse (no ischemia and reperfusion).

Figure 4B. Wild-type mouse after 90 minutes of warm ischemia, followed by 6
hours of reperfusion. There is signiﬁcant hepatocellular necrosis in the
pericentral and midzonal regions, with relative sparing of the periportal areas.

Note the presence of neutrophils in the midzonal region.

Figure 4C. P/l null mouse after 90 minutes of warm ischemia followed by 6 hours

of reperfusion. Again note the signiﬁcant hepatocellular necrosis in the

pericentral and midzonal regions, with relative sparing of the periportal areas.

47

 

 

48

Demonstration of Neutrophil Recruitment by Immunohistochemical
Staining

Immunohistochemical staining for neutrophils was utilized to assess
neutrophil recruitment. In situ immunohistochemical staining for neutrophils was
carried out using a speciﬁc mAb to mouse neutrophils. A scant number of
neutrophils were identiﬁed in the livers of sham operated wild-type and P/l null
mice at each time point (Figure 5A). However, after 90 minutes of ischemia and
6 hours of reperfusion a greater number of neutrophils inﬁltrated into the hepatic
parenchyma of wild-type mice, with the majority of neutrophils concentrated in
the pericentral and midzonal regions (Figure SB). Under a similar condition, P/l
null mice exhibited less neutrophil inﬁltration, with the majority of neutrophils
concentrated in the pericentral and periportal regions (Figure 5C). After 1.5 and
3 hours of reperfusion, there was minimal inﬁltration of neutrophils into the liver

parenchyma of both groups.

49

Figure 5A. Wild-type, 6 hour sham (No ischemia and reperfusion).

Figure 58. Wild-type mouse after 90 minutes of warm ischemia and 6 hours of

reperfusion.

Figure SC. P/l null mouse after 90 minutes of warm ischemia and 6 hours of

reperfusion.

50

 

 

51

DISCUSSION

The hypothesis of this study was P-selectin and lCAM-1 play an important
role in neutrophil-mediated hepatic IR injury. To test the hypothesis, the roles of
P-selectin and ICAM-1 in the pathogenesis of hepatic IR injury were investigated
collectively using mice deﬁcient in P-selectin and lCAM-1. Warm IR injury to the
liver leads to an acute Inﬂammatory response that may cause signiﬁcant cellular
damage and organ dysfunction. It is an important clinical problem that has been
demonstrated in a number of settings, including hepatic resection, liver
transplantation and hemorrhagic shock with ﬂuid resuscitation (Huguet et al.,
1994; Liu et al., 1996; Lemasters and Thruman 1997; Vedder et al., 1989).
Consequences of hepatic IR injury include liver failure and multiple organ
dysfunction syndrome, both of which have signiﬁcant rates of morbidity and
mortality (Lentsch et al., 2000). By focussing on P-selectin and ICAM-1, each
involved in the step-wise recruitment of neutrophils in hepatic injury, it was hoped
that the mechanism of this phenomenon-could be further elucidated and provide
a potential pathway to attenuate warm hepatic IR injury in a clinical arena.

Adhesion of neutrophils involves a multi-step process mediated by a
variety of cell surface molecules, with the selectins mediating the initial capture
and rolling of leukocytes along the endothelium, while the immunoglobulin gene
superfamily and integrins are involved in the subsequent ﬁrm adhesion and
transmigration. The fact that each class of adhesion molecules consists of
different members with similar functions raises the possibility of functional

redundancy within the system. Thus, while the double knockout mice were

52

deﬁcient in both P-selectin and ICAM-1, it is conceivable that neutrophil rolling
and transmigration may have been facilitated through a different combination of
adhesion molecules. Utilizing models of trauma and TNFor, Jung and Ley (1999)
concluded that overlapping functions were present among the selectin family in
mice lacking a combination of selectins and the elimination of E-, L- and P-
selectin signiﬁcantly impaired neutrophil recruitment. Furthermore, while it is
known that the recruitment of neutrophils requires the interaction between the (32-
integrins and lCAM-1, a recent study indicated that antibodies to VCAM-1 could
also inhibit liver injury by preventing neutrophil extravasation (Essani et al.,
1997). These results indicate that neutrophils may be able to utilize interactions
between B1-integrin and VCAM-1, as well as between B2-integrin and ICAM-1 for
transmigration.

To further support the concept of overlapping functions between adhesion
molecules, some investigators have challenged the paradigm of leukocyte
recruitment as a cascade of events occurring in separable, sequential steps that
are dependent upon different adhesion molecules. In TNFor-induced
inﬂammation, leukocyte rolling velocities in CD18 deﬁcient mice were signiﬁcantly
elevated, suggesting that BZ-integrins are involved in mediating leukocyte rolling
(Jung et al., 1998). Utilizing CD18 and E-selectin double mutant mice in a model
of TNFa-induced inﬂammation, Forlow et al (2000) demonstrated a signiﬁcant
increase in leukocyte rolling. These data indicate that the steps in leukocyte

recruitment are not discrete, independent events, but rather the functions of

53

selectins and integrins overlap to mediate a crucial step in the leukocyte
adhesion cascade.

Several studies have shown that blocking P—selectin and lCAM-1 adhesion
molecules with monoclonal antibodies or their ligands attenuates inﬂammatory
tissue injury following IR (Garcia-Criado et al., 1995; Nakano et al., 1995;
Marubayashi et al., 1997; Dulkanchainun et al., 1998; Singh et al., 1998; Yadav
et al., 1998). Administration of an antibody to P-selectin before and after
reperfusion, in models of partial or total hepatic ischemia has shown reduced
hepatic damage, decreased neutrophil inﬁltration and improved survival (Garcia-
Criado et al., 1995; Singh et al., 1998). Similarly, administration of soluble
PSGL-1 at the time of hepatic inﬂow occlusion in a warm ischemia model in the
rat has shown attenuated hepatic damage and neutrophil emigration
(Dulkanchainun et al., 1998). In addition the use of soluble PSGL-1 in the
University of Wisconsin cold preservation solution for organ transplantation,
signiﬁcantly increased the survival of rat liver isografts (Dulkanchainun et al.,
1998). Further, blocking lCAM-1 receptors with monoclonal antibodies has
resulted in hepatocellular protection with decreases in transaminase levels,
neutrophil inﬁltration and oxygen radical formation (Nakano et al., 1995;
Marubayashi et al., 1997).

Bullard et al (1995), who ﬁrst described P/l null double mutant mice, have
shown that while mice with either a P-selectin or lCAM-1 mutation alone showed
60-70% reduction in acute neutrophil emigration into the peritoneum during 8.

pneumoniae-induced peritonitis, double mutant mice showed a complete loss of

54

neutrophil emigration. In contrast, neutrophil migration into the alveolar spaces
during 8. pneumoniae-induced acute pneumonia was normal in double mutant
mice, indicating different pathways for neutrophil inﬁltration into various organs.
Examination of circulating leukocyte counts demonstrated a signiﬁcant
increase in the number of neutrophils in the PH null double mutants when
compared to wild-type mice of similar genetic background (Bullard et al., 1995).
This could certainly be considered a confounding variable in this investigation.
The exact mechanism for the increased circulating leukocyte counts in the Pll
null mice compared to wild-type mice is not known (Bullard et al., 1995). A
similar phenomenon has been observed in P-selectin deﬁcient mice (Mayadas et
al., 1993; Johnson et al., 1995). Mice deﬁcient in lCAM-1 have an even higher
elevation in circulating neutrophils (Sligh et al., 1993; Xu et al., 1994). In P-
selectin knockout mice, the higher level of circulating neutrophils was attributable
to a delay in neutrophil clearance, which suggests that P-selectin is important for
normal turnover of neutrophils in the circulation (Johnson et al., 1995). To
assess whether P-selectin deﬁcient mice and wild-type mice had comparable
marginated pools of neutrophils, Johnson et al. (1995) measured blood samples
before and after injection of epinephrine. Their results revealed that while the
ratios of response was higher in the wild-type mice relative to the P-selectin
knockout mice, the absolute number of neutrophils released into the circulation
was comparable. Therefore assuming a similar absolute neutrophil increase in
response to stress occurs in the P/l null mice compared to wild-type mice, then

the total number of circulating neutrophils available to respond to the hepatic HR

55

injury would be similar. Examination of neutrophil counts in P/l null mice
compared to wild-type mice before and after stimulation with epinephrine, as well
as with hepatic IR injury could provide some insight into the issue of neutrophilia.

The double knockout of the P/l null mice was conﬁrmed for both P-selectin
and lCAM-1, using reverse-transcriptase polymerase chain reaction and
immunohistochemical staining of the liver tissue, respectively on mice bred in
our laboratory. P-selectin immunohistochemistry was not performed. The
Jackson Laboratory conﬁrmed the PH null double mutation on mice purchased
directly from the company.

In this investigation a model of lobar hepatic ischemia was utilized.
lschemia was induced to the median and left lateral lobes, which results in
damage to approximately 70-80% of the liver mass (Kawano et al., 1989;
Martinez-Mier et al., 2000). Ninety minutes of partial hepatic ischemia followed
by various times of reperfusion caused a signiﬁcant hepatocellular injury in a
time-dependent manner In both wild-type and P/I null mice. The severity of the
hepatocellular damage between P/I null mice and wild-type mice was not
statistically signiﬁcant as judged by plasma ALT levels and liver histopathological
analysis. These results suggest that the hepatocellular injury induced following
reperfusion of the ischemic liver was independent of P-selectin and lCAM-1
molecules during the ﬁrst six hours of post-ischemic reperfusion. Furthermore,
IR injury sustained under these conditions may involve factors not directly related

to neutrophil inﬁltration.

56

Several investigations have observed two phases of liver injury following
hepatic IR (Colletti et al., 1990; Jaeschke et al., 1990). The ﬁrst or earlier phase
develops over the course of the ﬁrst one to three hours of reperfusion. Increased
levels of liver transaminases characterize this phase. It is mediated through an
ROS mechanism and correlates with Kupffer cell activation. The injury that
occurs during this phase is unrelated to intrahepatic neutrophils (Jaeschke et al.,
1991; Jaeschke et al., 1992). The second or later phase develops after six to
twenty-four hours of reperfusion. This is characterized by a further increase in
hepatic transaminases. It is also associated with a massive emigration of
neutrophils into the hepatic parenchyma (Jaeschke et al., 1993; Jaeschke et al.,
1993; Langdale et al., 1993; Suzuki et al., 1993). These two phases are
superimposed upon the damage that is initiated by the oxidative stresses that
occurs during ischemia. A limitation of this project was the reperfusion time
frame that was studied. Only the initial time point for neutrophil-mediated hepatic
IR injury was measured, which may explain the lack of difference observed
between the wild-type and P/l null mice. The project could be taken a step
further by looking at longer reperfusion times.

Several studies have used mice with double-gene targeted knockouts for
adhesion molecules. Kamochi et al (1999), have studied lipopolysaccharide
(LPS)—induced neutrophil recruitment and injury to the lung and liver. After
intraperitoneal injection of LPS, overall mortality was not reduced. However,
there was a signiﬁcant delay in mortality in the P/l null mice compared with that in

wild type mice (Kamochi et al., 1999). In addition, lung and liver edema was

57

signiﬁcantly lower in LPS-treated P-selectln/lCAM-1 null mice compared with
LPS-treated wild type mice. Similarly a study by Whalen et al., investigated the
inﬂammatory response after traumatic brain injury in P-selectin/lCAM-1 null mice
(Whalen et al., 2000). They observed no differences in brain neutrophil
accumulation between the wild type and the null mice. However, brain edema
was signiﬁcantly decreased in the null mice. In our study, we did not examine
liver edema formation in P/l null mice.

This investigation was limited by its design in that only double mutant mice
for P-selectin and lCAM-1 were studied. Applying the surgical model to mice that
are deﬁcient in either P—selectin or lCAM-1 may add signiﬁcant information
regarding a possible interaction between these two adhesion molecules.

A study by Yadav et al (1998) investigated the role of L-selectin and
lCAM-1 in hepatic I/R injury using the double mutant L-selectin/lCAM-1 null mice,
as well as single mutants. They observed that in livers subjected to 90 minutes
of ischemia followed by 1 hour of reperfusion, there was a signiﬁcant decrease in
inﬁltration of neutrophils between the null and the wild-type mice. However, after
6 hours of reperfusion, there was extensive inﬁltration of neutrophils in all areas
of the hepatic tissue, and no marked differences were detected between the null
and wild-type groups. Furthermore, mice subjected to 120 minutes of ischemia
with 6 hours of reperfusion had serum transaminase levels that were
paradoxically higher in the null mice compared with the wild-type.

An investigation that utilized P-selectin deﬁcient mice in a model of warm

partial hepatic ischemia, demonstrated an important role for P-selectin in

58

neutrophil and platelet sequestration (Yadav et al., 1999). Following 90 minutes
of ischemia, there was a signiﬁcant decrease in serum transaminase levels,
hepatocellular necrosis, as well as neutrophil inﬁltration and adhesion (Yadav et
al., 1999). In a model of total hepatic ischemia, there was improved survival in
mice subjected to both 75 and 90 minutes of ischemia (Yadav et al., 1999). In
addition, platelet sequestration, as determined by in situ immunohistochemical
staining, was markedly reduced in the P-selectin deﬁcient mice (Yadav et al.,
1999). In other studies using P-selectin knockout mice in a model of partial
hepatic ischemia, intravital microscopy was employed to investigate leukocyte
recruitment in the postischemic hepatic vasculature. An increase in rolling
leukocytes was observed in wild-type mice subjected to 20 minutes of ischemia
and 5 hours of reperfusion (Zibari et al., 1998). This phenomenon was not
observed in control mice, P—selectin deﬁcient mice, or wild-type mice treated with
a mAb to P-selectin (Zibari et al., 1998). Furthermore, when this same group
looked at the number of rolling, saltating and adherent leukocytes in terminal
hepatic venules after IR, there were signiﬁcant increases in wild-type mice,
whereas in P-selectin deﬁcient mice these responses were absent (Sawaya et
al., 1999). Together, these investigations point toward a role for P-selectin in
hepatic IR injury.

Some studies have concluded that P-selectin, in contrast to many other
non-hepatic tissues, is not required for sinusoidal neutrophil sequestration,
transendothelial migration, and subsequent neutrophil-induced liver parenchymal

injury. Since there are no Weibel-Palade bodies in sinusoidal endothelial cells of

59

normal liver (Wisse 1972; Irving et al., 1984), P-selectin expression in the liver
must be under transcriptional control (Essani et al., 1998). In a model of
endotoxemia, P-selectin mRNA formation was demonstrated in Kupffer cells as
well as endothelial cells (Essani et al., 1995). In a model of galactosamine and
Salmonella abortus equi endotoxemia, immunohistochemical analysis indicated
that P-selectin was expressed on endothelial cells of the periportal and central
vein areas. However P-selectin was not detected on sinusoidal endothelial cells
(Essani et al., 1998). In addition, administration of an anti-P-selectin mAb neither
reduced the number of accumulating neutrophils in the sinusoids, nor attenuated
liver injury. This study suggests that the lack of P-selectin expression on
sinusoidal cells and on hepatocytes makes a contribution of P-selectin to
transmigration and adherence to parenchymal cells unlikely.

The results of these investigations by Essani and Jaeschke lend support
to a three-step mechanism of neutrophil-mediated parenchymal injury: 1)
neutrophil sequestration in the hepatic vasculature, 2) transendothelial migration,
and 3) adherence to hepatocytes which leads to the release of cytotoxic
mediators (Jaeschke 1997). The ﬁrst step is sequestration of activated
neutrophils in the hepatic vasculature. Even though neutrophils can be found in
sinusoids and marginated in post-sinusoidal venules, only cells in sinusoids
appear to be relevant for injury (Chosay et al., 1997). The trapping hypothesis of
neutrophil sequestration requires these cells to be mechanically trapped in the
liver vasculature by swelling of the endothelial lining cells (McCuskey 1993),

active sinusoidal constriction (Zhang et al., 1994), and decreased deformability of

60

neutrophil membranes (Worthen et al., 1989). The absence of P-selectin
expression demonstrated by immunohistochemistry in a model of endotoxemia
suggests that P-selectin is not involved in this initial inﬂammatory response.
After the initial sequestration, neutrophils can transmigrate out of the vasculature
into the liver parenchyma, adhere to the hepatocytes, and induce adherence-
dependent cytotoxicity (Jaeshke 1997).

A potential shortcoming of this surgical model is the prolonged duration of
ischemia. While another study has utilized this model successfully, identiﬁcation
of a critical ischemia time that results in the obstruction of sinusoids by blood
elements has not been identiﬁed. The contribution of adhesion molecules in
mediating microcirculatory disturbances due to Ieukostasis has remained
controversial. While some authors have proposed that the development of
microcirculatory occlusion is solely dependent upon factors such as endothelial
swelling, protrusion of blebs, and hemoconcentration (Menger et al., 1993),
others have suggested that neutrophil plugging is an important event in the
occlusion of microvasculature after reperfusion (Ferguson et al., 1993; Koo et al.,
1992). This phenomenon of no-reﬂow has been described to occur as early as
after 30 minutes of hepatic ischemia (Koo et al., 1992). Intravital microscopy
could be used to determine the ischemia time point at which microvascular failure
occurs in this model. Yadav et al (1998) utilized this technique to establish a
critical ischemia time in a model of murine partial hepatic ischemia where 39% of
the liver mass was affected. After 60 minutes of ischemia, microcirculatory

failure was manifested as petechial bleeding. In livers subjected to 90 and 120

61

minutes of ischemia, there was extensive clot formation and vascular plugging
(Yadav et al., 1998). It is possible that the lack of signiﬁcant difference in ALT
levels observed between the wild-type and P/l null mice may be secondary to
poor perfusion of liver tissue with the inability to completely release enzymes in
the circulation.

In situations where there is marked damage to sinusoidal endothelial cells,
transmigration may not be a signiﬁcant factor in the pathophysiology of
neutrophil-induced hepatic parenchymal injury. This may occur during severe IR
injury (McKeown et al., 1988; Caldwell-Kenkel et al., 1989; Fisher et al., 1997).
Under these conditions, neutrophils may have direct access to parenchymal cells
without undergoing transmigration. This may explain the neutrophil inﬁltration
seen in both wild-type and P/l null mice after 90 minutes of ischemia and 6 hours
of reperfusion. Furthermore, this may also explain the differential effect of
adhesion molecules in various models of inﬂammation. As an example, an
lCAM-1 antibody was found to be highly effective in attenuating liver injury due to
the prevention of neutrophil transmigration during endotoxemia (Essani et al.,
1995). Yet, a similar antibody was signiﬁcantly less effective in preventing liver
injury during hepatic IR (Farhood et al., 1995).

lschemic injury of the liver has been generally considered to result in
necrosis. However, it has been recently recognized that mediators of apoptosis
are activated during IR injury in both sinusoidal endothelial cells and
hepatocytes. In a study of normothermic IR injury, sinusoidal endothelial cells

showed evidence of apoptosis earlier than hepatocytes (Kohli et al., 2000). With

62

a longer duration of ischemia, a greater number of sinusoidal endothelial cells
and hepatocytes undenNent apoptosis. This was also observed with increasing
duration of reperfusion. These results suggest that apoptosis of endothelial cells
followed by hepatocytes is an important mechanism of cell death after IR injury in
the liver. In support of this suggestion a study Cursio et al (1999), has shown
that inhibition of liver apoptosis by applying a capsase inhibitor fully protected
rats from death induced by normothermic hepatic IR. In addition, a capsase
inhibitor reduced sinusoidal endothelial cell apoptosis and improved survival in a
model of orthotopic liver transplantation (Natori et al., 1999). An investigation
that utilized an in vivo adenovirus-mediated gene transfer of the antiapoptotic
gene Bel-2, demonstrated a signiﬁcant decrease in hepatic IR injury based on
transaminases, necrosis and apoptosis, and permanent survival (Bilbao et al.,
1999). Collectively these studies further support the hypothesis that severe IR
injury could damage the sinusoidal endothelial cells to such an extent that
adhesion-dependent transendothelial migration may not be a crucial step in
neutrophil-induced hepatic parenchymal injury.

The study presented here demonstrated that there was signiﬁcant
neutrophil inﬁltration after 6 hours of reperfusion in both wild-type and P/l null
mice seen on histopathology and immunohistochemistry. Qualitatively, there
was slightly less hepatic neutrophil inﬁltration in P/l null mice compared to wild-
type mice. This result could be further quantiﬁed either by formal neutrophil
counts from the ﬁxed slides or by measuring myeloperoxidase activity as

described by Mullane et al (Mullane et al., 1985; Bradley et al., 1982).

63

The presented study and the published studies collectively suggest that
murine regulation of adhesion pathways differs during lifelong deﬁciency of P-
selectin and lCAM-1 compared with inhibition of the adhesion molecules by
antibodies. In addition, the studies indicate that adhesion-dependent and
-independent activation of neutrophil emigration exists and can be differentially
regulated by the targeted tissues.

In conclusion, the presented study has shown hepatocellular injury
following IR in double P-selectin/ICAM-1 deﬁcient mice. The study suggests that
P-selectin and lCAM-1 adhesion molecules may not be critical factors in
hepatocellular injury induced by IR during the ﬁrst six hours of post-ischemic

reperfusion.

64

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