Intercellular Adhesion Molecule-1 and CD18 Are Involved in
Neutrophil Adhesion and Its Cytotoxicity to Cultured Sinusoidal
Endothelial Cells in Rats
SHINICHI SAKAMOTO,1 TAKESHI OKANOUE,1 YOSHITO ITOH,1 KYOKO SAKAMOTO,2 KENICHI NISHIOJI,1 YOSHIO NAKAGAWA,1
NORIMASA YOSHIDA,2 TOSHIKAZU YOSHIKAWA,2 AND KEI KASHIMA1

The expression of several adhesion molecules is increasedon the hepatic sinusoidal endothelial cells (SECs) in various
liver diseases. The objective of this study is to assess the rolesof intercellular adhesion molecule 1 (ICAM-1) and of CD18 in
the interaction between the neutrophils (polymorphonuclear
leukocytes [PMNs]) and SECs and in the injury to SECs mediated by PMNs. Rat PMNs was perfused on SECs stimulatedwith tumor necrosis factor a (TNF-a) using an in vitro flow
system. The number of adhered PMNs to SECs and that of
PMNs migrated under SECs was counted and the effects ofanti–ICAM-1, anti–CD18, and dexamethasone were studied.
We also define the effect of these antibodies on the SEC injury
mediated by PMNs stimulated with phorbol 12-myristate 13acetate (PMA) or N-formyl-methionyl-leucyl-phenylalanine(fMLP). TNF-a significantly increased the adhesion of PMNs
to SECs (322 { 26 cells/mm2) compared with controls (194{ 22 cells/mm2). Anti–ICAM-1 and anti–CD18 significantly
inhibited the adhesion of PMNs (131 { 10 and 51 { 30cells/mm2, respectively). These antibodies also decreased themigration rate of PMNs (6.0% and 7.9%, respectively) compared with controls (migration rate, 21.2%). The SEC injury
induced by PMA- and fMLP-activated PMNs was prevented
by anti–ICAM-1 and anti–CD18. The adhesion of PMNs induced by TNF-a was inhibited by the treatment with dexamethasone (160 { 20 cells/mm2) via a down-regulation of
ICAM-1 expression on SECs. The interactions between ICAM1 and CD18 appeared to be important in the adhesion andthe migration of PMNs to SECs. The injury to SECs was
induced by the close interaction between the activated PMNs
and SECs mediated via ICAM-1 and CD18. (HEPATOLOGY
1997;26:658-663.)

Abbreviations: ICAM-1, intercellular adhesion molecule 1; PMN, polymorphonuclear leukocyte; SEC, sinusoidal endothelial cell; TNF-a, tumor necrosis factor a;
HBSS, Hank’s balanced salt solution; ELISA, enzyme-linked immunosorbent assay;
PMA, phorbol 12-myristate 13-acetate; fMLP, N-formyl-methionyl-leucyl-phenylalanine.
From the 1Third Department of Internal Medicine; and the 2First Department ofInternal Medicine, Kyoto Prefectural University of Medicine, Kyoto, Japan.
Received March 12, 1997; accepted April 10, 1997.
Supported in part by grant-in-aid 07457139 from the Ministry of Education, Science
and Culture of Japan.
Presented in part at the 1995 Annual Meeting of the American Association for the
Study of Liver Disease in Chicago, IL.
Address reprint requests to: Takeshi Okanoue, M.D., Third Department of Internal
Medicine, Kyoto Prefectural University of Medicine, Kawaramachi-Hirokoji, Kamigyoku, Kyoto 602, Japan. Fax: 81-75-251-0710.
Copyright q 1997 by the American Association for the Study of Liver Diseases.
0270-9139/97/2603-0019$3.00/0

Leukocytes must interact with endothelial cells before they
can infiltrate the tissue. Specific interactions between theadhesion molecules on the surface membranes of leukocytes
and endothelial cells mediate the recruitment of leukocytes
into inflammatory sites.1 Intercellular adhesion molecule-1(ICAM-1), a member of the immunoglobulin family, is reported to be expressed on endothelial cells and to bind toleukocyte integrins such as Mac-1 (CD11b/CD18) on theneutrophils (polymorphonuclear leukocytes [PMNs])2 or
lymphocyte function-associated antigen 1 (CD11a/CD18) on
the lymphocytes.3 ICAM-1 is present at the basal and luminalsides of endothelial cells and plays a key role in the strong
adhesion, as well as the transendothelial migration of human
T lymphocytes and PMNs.4,5 For these local interactions between leukocytes and endothelium, the localized calcium
flux is induced in endothelial cells surrounding the intruding
leukocyte.6 Integrin-associated protein (CD47) is involved incalcium regulation in endothelium to be essential for neutrophil migration through endothelium.7
Immunohistochemical study has shown these adhesion
molecules to be involved in the pathogenesis of various liverdiseases. Volpes et al.8 observed that the expression of ICAM1 was increased on the sinusoidal endothelial cells (SECs)
in inflamed lesions that lymphocyte function-associated antigen 1-positive lymphocytes infiltrated. Burra et al.9 statedthat the increased expression of ICAM-1 and of vascularendothelial adhesion molecule 1 on the hepatic SECs of patients with alcoholic liver cirrhosis might cause hepatic injury. Thus far, there are few studies on the interaction between PMNs and SECs in vitro.
Large numbers of PMNs accumulate in the liver tissueof patients with alcoholic hepatitis and septic liver injury
associated with the development of liver injury.10,11 Komatsu
et al.12 showed that the depletion of PMNs by use of antineutrophil antibody prevents liver dysfunction in rats withsepsis. These reports suggest that PMNs are important in thepathogenesis of such disorders. The present study addresses
the hypothesis that an ICAM-1-CD18-mediated interaction
may be involved in the adhesion of PMNs to SECs in PMNmediated liver injury. We have used an in vitro flow systemthat allows for the dynamic analysis of the spatial and temporal details of cell-to-cell interactions13,14 and have assessed
the interaction between PMNs and SECs stimulated withtumor necrosis factor a (TNF-a).
Endothelial cell injury elicited by activated leukocytes isknown to be the initial stage of various inflammatory diseases. The adhesion of leukocytes to the endothelium has

658

AID

Hepa 0027/

5p26$$$521

08-13-97 09:26:48

hepa

WBS: Hepatology

HEPATOLOGY Vol. 26, No. 3, 1997

SAKAMOTO ET AL.

659

been recognized as an important process in leukocytes causing endothelial cell injury.15 This injury is mainly caused by
the active oxygen species or several proteases released by
activated leukocytes.16,17 Then, we investigated the role ofadhesion molecules, radical scavengers, and a protease inhibitor on PMN-mediated injury to SECs. Additionally, we evaluated the interaction between PMNs and SECs and theexpression of ICAM-1 on SECs in the presence of dexamethasone because glucocorticoids are administered to patientswith alcoholic hepatitis.
MATERIALS AND METHODS
Animals. Male Wistar rats (Shimizu Animal, Inc., Kyoto, Japan)(weight range, 200-250 g) were used in this study. They were fed
a standard laboratory diet and water ad libitum under standard
laboratory conditions. Procedures were performed according to theguidelines of the ‘‘Guide for the Care and Use of Laboratory Animals’’ from the National Institutes of Health.
Isolation and Culture of Rat SECs. SECs were obtained from male
Wistar rats according to a previously described method18 with aslight modification. Briefly, the liver was perfused via the portal
vein with Hank’s solution that was free of Ca// and Mg// andcontained 0.01% ethylenediaminetetraacetic acid and 10 U/mL ofheparin. The liver was perfused subsequently with Hank’s balanced
salt solution (HBSS) supplemented with 0.05% collagenase. The
liver was then removed, cut into pieces, and passed through a meshto remove cell debris. Low-speed centrifugation was performed
twice to separate the hepatocytes. The suspension of nonparenchymal cells was then introduced into a centrifugal elutriation rotor(Hitachi Koki Co., Ltd., Ibaragi, Japan) to isolate the SECs. SECs
were suspended in RPMI 1640 medium (Nissui Pharmaceutical Co.,
Ltd., Tokyo, Japan) containing 10% fetal calf serum (GIBCO Laboratories, Grand Island, NY), 1007 mol/L dexamethasone (Sigma
Chemical Co., St. Louis, MO), and 0.1% gentamycin sulfate (Sigma
Chemical Co.) at a density of 2 1 106 cells/mL. An aliquot (1.0mL) of the endothelial cell suspension was cultured on a 24 1 50mm glass coverslip (Matsunami Glass Industries, Ltd., Osaka, Japan) and coated with type I C collagen (Nitta Gelatin, Inc., Osaka,
Japan) for 2 hours at 377C in 5% CO2 . Coverslips were then rinsed
and cultured in a medium free of dexamethasone for another 10hours for the adhesion assay. To measure the expression of ICAM1 by enzyme-linked immunosorbent assay (ELISA), 100 mL of theendothelial cell suspension (2 1 1005 cells/well) was cultured on
a 96-well plate coated with type I C collagen (Corning 25860 CPL
1; Iwaki Glass, Tokyo, Japan) as described previously. SECs wereidentified morphologically by scanning electron microscopy andhistochemically by use of DiI-acetylated low-density lipoproteinlabeled with the fluorescent probe.19 About 92% of cultured endothelial cell fraction showed the characteristic features of pores andsieve plates and took up acetylated low-density lipoprotein. Immunohistochemical staining for anti-rat monocyte/macrophage antibody (ED1; Chemicon International Inc.,Temacula, CA) showedthat 4% to 6% of cultured endothelial cell fraction contained liver
macrophages.
Isolation of Rat PMNs. Rat PMNs were isolated from the peritoneal
fluid 4 hours after the intraperitoneal injection of 15 mL of 0.1%
glycogen obtained from oysters (Sigma Chemical Co.). After washing with HBSS, the cells were suspended in HBSS at a density of 1
1 106 cells/mL and maintained at 47C for 1 hour. Giemsa staining
showed ú95% of the isolated cells to be PMNs. Flow-cytometricalanalysis showed that 96% of the cell fraction isolated from theperitoneal fluid expressed CD18 (WT.3; Seikagaku Corp., Tokyo,
Japan) on their own surface (data not shown) as previously reported.20
Adhesion Assay. The adhesion and migration of PMNs on SECs
were assessed using an in vitro flow system.13,14 The chamber consisted of a collagen-coated coverslip with a confluent monolayer of
SECs that was attached to a polycarbonate base. The space between

AID

Hepa 0027/

5p26$$$521

08-13-97 09:26:48

FIG. 1. Phase-contrast micrographs of adhesion and migration of neutrophils to monolayer of hepatic SECs. A is focused on the luminal surfaceof the monolayer. B is focused just beneath the monolayer. The neutrophils
adherent on the monolayer (arrow) were rounded up and appeared as brightobjects, and those migrated to the subluminal space (arrowhead) became
spread and phase-dark. (Original magnification 1200.)

the coverslip and the base was held approximately 250 mm apartby a silastic rubber gasket. The flow rate was controlled with asyringe pump (Iwaki Glass Co., Ltd.). Suspensions of PMNs werepassed through the chamber at a wall shear stress of 0.5 dyne/cm2
and 377C. Shear stress, t in dyne/cm2, was calculated from thefollowing equation:t Å 3m Q/2ba2,
where t is the wall shear stress, m is the viscosity of water at 377C,
which is used as an approximation of the viscosity of HBSS (0.007
poise), Q is volumetric flow rate, a is the half channel height, andb is the channel width. The wall shear stress in the hepatic sinusoid
was calculated from leukocyte velocity and the diameter of the
microvessels that were measured by intravital fluorescence microscopy used by Vollmar et al.21 The wall shear stress in the hepatic
sinusoid was lower than that in venule because of its slow blood
flow. A wall shear stress of 0.5 dyne/cm2 is supposed to be within
pathophysiological condition in hepatic sinusoids, based on theirfindings. SECs on the coverslips were stimulated with 100 U/mL ofrecombinant human TNF-a (rhTNF-a) (Dainippon PharmaceuticalCo., Osaka, Japan) for 4 hours, mounted in flow chambers, andperfused for 1 minute with HBSS (rinse step) followed by an 8minute perfusion with a suspension of PMNs (perfusion step). In
some experiments, an antibody against human TNF-a (Dainippon
Pharmaceutical Co.) was added. The interaction between PMNs and
SECs was observed by phase-contrast microscopy (Olympus Co.,
Ltd., Tokyo, Japan) and was recorded on a videotape. The numberof PMNs that remained in contact with the endothelial cell monolayer was determined in six digitized frames of different fields ofview from the last minute (7-8 minute) of the perfusion step andwas expressed as the number of cells that adhered or migrated
per square millimeter. The PMNs that adhered to the monolayer
possessed spherical geometry and appeared as phase-bright objects,
whereas those that migrated beneath the monolayer were flat andappeared as phase-dark objects, as described previously14 (Fig. 1).
The migration rate was quantitated as follows:
Migration rate (%)Å Migrated PMNs 1 100 4 (Adhered PMNs / Migrated PMNs).
In some experiments, pretreatment of SECs with anti-rat ICAM-

hepa

WBS: Hepatology

660 SAKAMOTO ET AL.

HEPATOLOGY September 1997

added to each well and incubated for 6 hours in the presence orabsence of the following: anti–ICAM-1 antibody, anti-CD18 antibody, catalase (1,000 U/mL) (Sigma Chemical Co.), superoxide
dismutase (1,000 U/mL) (Seikagaku Corp.), and aprotinin (1,000
U/mL) (Sigma Chemical Co.). In some experiments, 2 1 104 cells
or 5 1 104 cells of PMNs were added to the well (15 PMN groupand 12 PMN group, respectively). 51Cr Radioactivity released fromthe injured SECs was measured. The percentage of specific 51Cr
release, an index of injury to SECs, was quantitated as follows:% Specific 51Cr ReleaseÅ (Experimental Count — Spontaneous Count) 1 100
4 (Total Count — Spontaneous Count).
The total count was taken as that for the well receiving 0.2%
Triton X-100 containing 0.1N NaOH and spontaneous count forthat with culture medium only. The experimental count represents
the count for 51Cr release induced by PMA- or fMLP-stimulated
PMNs with or without reagents.
Statistical Analysis. Values are expressed as means { SEM. Data
were analyzed by analysis of variance and Student’s t test, with
Bonferroni’s correction for multiple comparisons. An unpaired twotailed Student’s t test was used to compare the migration rates andMigration Rate (%)
percent inhibitions. A P value of õ.05 was considered statisticallyÅ Migrated PMNs 1 100 4 (Adhered PMNs / Migrated PMNs). significant.
FIG. 2. Effects of monoclonal antibodies to ICAM-1 or CD18 and dexamethasone (DEX; 1 1 1007 mol/L) and anti–TNF-a on neutrophil adhesion
and migration to SECs induced by rhTNF-a (100 U/mL for 4 hours). Monoclonal antibodies were added at a final concentration of 5 mg/mL. Each value
represents means { SEM of three experiments performed in duplicate. The
percent shows the migration rate of neutrophils determined by the following
equation:#P õ .05 compared with control. *P õ .05 compared with 100 U/mL TNFa group without monoclonal antibody or dexamethasone.

RESULTS
Adhesion and Migration of PMNs to TNF-a –Stimulated SECs.

1 monoclonal antibody (1A29; 5 mg/mL, Seikagaku Corp.) or pretreatment of isolated PMNs with anti-rat CD18 (5 mg/mL) for 20
minutes at 377C was performed.
Expression of ICAM-1 on SECs. The expression of ICAM-1 on SECs
was measured by cellular ELISA. SECs cultured on a 96-well plate
coated with type I C collagen were incubated with 1, 10, 100, and
1,000 U/mL of rhTNF-a for 4 hours and fixed in 4% paraformaldehyde. In some experiments, an antibody against human TNF-a
was added. To investigate the effect of dexamethasone on ICAM-1
expression, serial concentrations of dexamethasone were added insome experiments. Three washes were done in phosphate-bufferedsaline containing 0.1% bovine serum albumin. Nonspecific reactions were blocked by incubation for 30 minutes with 1% normal
goat serum (Sigma Chemical Co.). After an additional wash, cells
were incubated for 1 hour with 50 mL of anti-rat ICAM-1 monoclonal antibody at a dilution of 1:1000. After three washes withphosphate-buffered saline that contained bovine serum albumin, thecells were incubated for 1 hour with a peroxidase-labeled antibody
against mouse immunoglobulin G (Seikagaku Corp.). Peroxidase
staining was then performed with o-phenylenediamine (Sigma
Chemical Co.) and H2O2 . Absorbance at 490 nm was measured by
an Immunoreader (Multiskan; Labosystems, Helsinki, Finland). The
microplates were subsequently washed in tap water before 50 mL
of 0.5% crystal violet was added for 5 minutes. After a thoroughwash in tap water, 100 mL of 33% acetic acid was used to solubilize
the nuclear stain, and absorbance at 570 nm was measured, representing the actual cell count per well. The expression of ICAM-1
was provided as the ratio between specific protein measurement
and cell density.
Assay of Injury to the SECs. 51Cr Release was used as an index of
the injury of SECs. This method has been used for the assay of cellor chemical-mediated cytotoxicity.22,23 In brief, SECs were seeded
into a 48-well culture plate coated with type I C collagen and wereincubated with culture medium containing 15 mCi Na51CrO4 /mL
for 12 hours. SECs were washed twice with culture medium toremove unincorporated radioactivity. After stimulating the SECs
with rhTNF-a (100 U/mL for 4 hours), 1 1 105 cells of rat PMNs
in a culture medium containing 10 ng/mL of phorbol 12-myristate
13-acetate (PMA) (Sigma Chemical Co.) or 100 nmol/L of N-formylmethionyl-leucyl-phenylalanine (fMPL) (Sigma Chemical Co.) was

AID

Hepa 0027/

5p26$$$521

08-13-97 09:26:48

The number of adherent PMNs to the monolayers of SECs
was increased significantly by stimulation with 100 U/mL of
rhTNF-a (Fig. 2). This was inhibited by the addition ofneutralizing anti–TNF-a antibody. To investigate the effects
of ICAM-1 and CD18 on the adhesion of PMNs, we assessed
the adhesion of PMNs after pretreating the SECs with antirat ICAM-1 antibody or after pretreating the PMNs with antiCD18 antibody. Five micrograms per milliliter each of theanti–ICAM-1 and anti-CD18 antibody significantly reduced
the number of adherent PMNs compared with that observedin the absence of an antibody (Fig. 2). Each antibody significantly reduced the migration rate of PMNs (P õ .05).
Effect of TNF-a on ICAM-1 Expression. To determine whether
the increase in PMN adherence induced by rhTNF-a wasrelated to an increased expression of ICAM-1, we determinedthe expression of ICAM-1 on SECs by cellular ELISA. TNFa enhanced the expression of ICAM-1 on SECs in a dosedependent manner. The presence of anti–TNF-a antibody
inhibited this effect (Fig. 3).

FIG. 3. Effects of rhTNF-a on the expression of ICAM-1 on SECs.
ICAM-1 expression on SECs stimulated with rhTNF-a for 4 hours wasmeasured by ELISA. Each value represents means { SEM of three experiments performed in triplicate. #P õ .05 compared with control.

hepa

WBS: Hepatology

HEPATOLOGY Vol. 26, No. 3, 1997

SAKAMOTO ET AL.

PMN-Mediated Injury to SECs. The injury to SECs caused by
PMA- or fMLP-stimulated PMNs are shown in Fig. 4. Culture
with the PMA- or fMLP-stimulated PMNs induced the releaseof 51Cr from SECs. The reduction in the number of cocultured PMNs resulted in the decrease of this endothelial cytotoxicity. Anti–ICAM-1 and anti-CD18 antibody each suppressed both PMA- and fMLP-induced injury to SECs. The
addition of catalase, which breaks down hydrogen peroxide,
suppressed PMA-induced injury to SECs but not fMLP-induced injury to SECs. The addition of superoxide dismutase,which breaks down superoxide anion, had little effect on
both SEC injuries. The cytotoxicity by fMLP-stimulated
PMNs was significantly abrogated by the addition of 1,000
U/mL of aprotinin, a protease inhibitor.
Effects of Dexamethasone on the Expression of ICAM-1 and on
PMN and SEC Interactions. To investigate the anti-inflamma-

tory mechanism of dexamethasone, we assessed its effect on
the expression of ICAM-1 expression on SECs. Dexamethasone suppressed the overexpression of ICAM-1 induced by
rhTNF-a dose-dependently (Fig. 5). Concentrations of dexamethasone of ¢1 1 1008 mol/L showed a significant effect.
As shown in Fig. 1, 1 1 1007 mol/L of dexamethasone significantly inhibited the adhesion of PMNs to SECs. Although the661

FIG. 5. Effects of dexamethasone (DEX) on ICAM-1 expression on
SECs. ICAM-1 expression was assessed by ELISA on TNF-a –stimulated
sinusoidal endothelial cells in the presence of serial concentrations of dexamethasone. Each value represents means { SEM of three experiments performed in triplicate. #P õ .05 compared with 100 U/mL TNF-a group
without dexamethasone.

migration rate of PMNs tended to decrease in the presenceof 1 1 1007 mol/L of dexamethasone, the effect was not
significant.
DISCUSSION

FIG. 4. Role of adhesion and neutrophil-derived oxidants in SEC injury
induced by PMNs. 51Cr Release was assessed at 6 hours after incubation.
Each value represents means { SEM of three experiments performed intriplicate and is expressed as the percentage of the control. The control
represents cytotoxicity induced by 1 1 105 cells of rat PMNs. The 51 PMN
and the 21 PMN represent cytotoxicity induced by 2 1 104 cells and 5 1 104
cells of PMNs, respectively. #P õ .05 compared with PMN group (control)and ##P õ .01 compared with PMN group.

AID

Hepa 0027/

5p26$$$521

08-13-97 09:26:48

Although numerous PMNs infiltrate the liver including
the hepatic sinusoids in alcoholic- or sepsis-related liver injury, the mechanism is not well understood. To clarify themechanism of the liver injury mediated by PMNs, we focusedon the interaction between ICAM-1 and CD18. This study
showed that the interaction between ICAM-1 and CD18 was
involved in PMN adhesion to and migration through SECs
and that this interaction was important for the injury to SECs
induced by PMA- and fMLP-activated PMNs.
The role of the adhesion molecules in the interaction between the leukocytes and endothelial cells was pathophysiologically investigated in vivo by using an intravital microscopein the mesentric circulation of rats24 and in vitro by using
human umbilical vein endothelial cells in an in vitro adhesion
assay.13,14 These experiments showed that ICAM-1 is important for the adhesion of PMNs to the endothelium.
CD11b/CD18 monoclonal antibody is reported to prevent thehepatic infiltration of PMNs and the necrosis of hepatocytes
induced by the intraperitoneal injection of endotoxin.25 This
study suggests that an ICAM-1-CD11b/CD18–mediated interaction is important for the accumulation of PMNs in theliver to induce hepatocellular necrosis.
We investigated the roles of ICAM-1 and CD18 in theadhesion of PMNs to SECs and the migration of PMNs
through SECs by means of an in vitro flow system. Thissystem provides for the dynamic analysis of the interactions
between the PMNs and SECs, such as rolling, adhesion, andmigration. We showed that the pretreatment of SECs with
rhTNF-a induced the adhesion of PMNs and that the anti–
ICAM-1 and anti-CD18 antibody each inhibited the increasein PMN adhesion (Fig. 2). Each antibody also prevented themigration of PMNs beneath the SEC monolayer. These results are consistent with those of Issekutz et al.,26 whoshowed that the transendothelial migration of PMNs beneatha human umbilical vein endothelial cell monolayer was
blocked by ú90% by anti-CD18 and partially blocked byanti–ICAM-1. Our results indicate that ICAM-1 and CD18

hepa

WBS: Hepatology

662 SAKAMOTO ET AL.

HEPATOLOGY September 1997

both play a key role in the adhesion and the migration of
PMNs in the hepatic sinusoids.
It is reported that ICAM-1 is weakly expressed on SECs ofnormal rat in vivo.27 Our cellular ELISA showed that ICAM-1
was expressed on SECs in basal conditions in vitro and thatrhTNF-a increased its expression in a dose-dependent manner. Ohira et al. reported that serum levels of TNF-a and
ICAM-1 expression on SECs were increased in rats treatedwith lipopolysaccharide.28 Our in vitro study supports theirobservation that TNF-a might induce PMN infiltration in
hepatic sinusoids by the induction of ICAM-1 on SECs.
The injury to the endothelial cells elicited by activatedleukocytes is the initial event in various forms of inflammation. It is mainly caused by the active oxygen species orseveral proteases released by activated PMNs.16,17,29 Komatsu
et al. reported that the serum from rat treated with galactosamine-lipopolysaccharide enhanced the release of oxygenderived free radicals by PMNs that contribute to the hepatic
injury.12 We showed that both PMA- and fMLP-activatedPMNs induced SEC injury in a PMN/SEC ratio–dependent
manner. This shows that PMNs infiltrating hepatic sinusoids
could induce the endothelial cell damage that causes thedisturbance of sinusoidal microcirculation.
Weiss has stated that oxygen radicals and proteases cooperate or interact in the tissue damage caused by PMNs.16 To
evaluate the role of the active oxygen species or proteases in
PMA- or fMLP-induced SEC injury, assays were performedin the presence of reactive oxygen scavengers or a proteaseinhibitor. In the present study, we showed that catalase prevented PMA-induced SEC injury, which seemed to be induced by hydrogen peroxide but not fMLP-induced SEC injury. This finding is supported by Fujita et al.30 who showedthat PMA-stimulated PMNs injure human umbilical vein endothelial cells by producing hydrogen peroxide and by inducing the increase in the level of intercellular peroxides inendothelial cells. In addition, we showed that fMLP-induced
PMN-mediated SEC cytotoxicity was inhibited by aprotinin
but not by reactive oxygen scavengers. Aprotinin is a broadspectrum protease inhibitor that has been used in the management of experimental endotoxin and septic shock.31 This
showed that fMLP-stimulated PMNs cause SEC injury mainly
by release of PMN protease. Endotoxin-related liver injury
involves SEC injury caused by infiltrated PMNs28 and is inhibited by protease inhibitors.32 Our in vitro study showedthat SEC injury caused by activated PMNs involved active
oxygen species or proteases.
The present study also showed that anti–ICAM-1 and antiCD18 each protected against SEC injury induced by both
PMA- and fMLP-activated PMNs, showing that these adhesion molecules were crucial for SEC injury mediated by different mechanisms, the active oxygen species and proteases.
It is reported that activated neutrophils produce hydrogen
peroxide and that the CD18-mediated interaction with endothelial cells increases hydrogen peroxide production by neutrophils.33 The treatment with anti–ICAM-1 or CD18 antibody not only inhibited the close contact between SECs andneutrophils but also might decrease the production of active
oxygen species. Injury to SECs leads to vascular protein leakage and to an increase in the extravasation of PMNs. Migrated
PMNs achieve access to the hepatocytes. Inflammatory cytokines promote the expression of ICAM-1 on the hepatocytes
and increase the adhesion of the lymphocytes to the hepatocytes.34 Essani et al.35 reported that anti–ICAM-1 reduced

AID

Hepa 0027/

5p26$$$521

08-13-97 09:26:48

extravasation of PMNs to hepatocytes and inhibited liver
injury. This is consistent with our finding that anti–ICAM1 reduced the migration of PMNs beneath the SEC monolayer. The protective effect of anti–ICAM-1 against thehepatic injury induced by PMNs may be explained by our
results showing that anti–ICAM-1 inhibited the close contact
between the PMNs and SECs and reduced the injury to SECs
induced by activated PMNs.
The involvement of leukocyte adhesion in the pathogenesis of hepatic injury related to alcoholism or sepsis has clinical relevance. It is reported that corticosteroids improve thesurvival in patients with severe alcoholic hepatitis36; however, its mechanism has not been clarified yet. We investigated the effects of dexamethasone on TNF-a –induced overexpression of ICAM-1 on SECs and on the interaction
between PMNs and SECs to clarify the mechanism of theanti-inflammatory effects of dexamethasone in PMN-mediated liver injury. Dexamethasone inhibited the expression of
ICAM-1 on SECs in a dose-dependent manner. The effects
of dexamethasone at concentrations of ú1 1 1008 mol/L
were statistically significant. In an in vitro adhesion assay, at
a concentration of 1 1 1007 mol/L, dexamethasone significantly inhibited the adhesion to SECs and tended to reducethe migration rate of PMNs. During rejection of liver allografts, corticosteroids reduce the expression of ICAM-1 on
hepatic endothelium and hepatocytes and prevent the rejection.37 Our findings support the hypothesis that corticosteroids prevent the hepatic accumulation of PMNs, in part, by
down-regulating ICAM-1 on SECs.
In summary, the interaction between ICAM-1 and CD18
was involved in the adhesion and migration of PMNs inhepatic sinusoids mediated by TNF-a. This interaction was
the key to the SEC injury induced by activated PMNs. Dexamethasone inhibited the adhesion of PMNs to SECs by reducing the expression of ICAM-1 on SECs. Blocking the interaction of ICAM-1-CD18 may point to a therapeutic strategy
for preventing liver injury mediated by PMNs.
Acknowledgment: The authors thank Nanako Kaibara forher excellent technical assistance in this study.
REFERENCES
1. Butcher EC. Leukocyte-endothelial cell recognition: three (or more)
steps to specificity and diversity. Cell 1991;67:1033-1036.
2. Diamond MS, Staunton DE, De Fougerrolles AR, Stacker SA, GarciaAguilar J, Hibbs ML, Springer TA. ICAM-1 (CD54): a counterreceptor
for Mac-1 (CD11b/CD18). J Cell Biol 1990;111:3129-3139.
3. Dustin ML, Springer TA. Lymphocyte function-associated antigen-1(LFA-1) interaction with intercellular adhesion molecule-1 (ICAM-1)
is one of at least three mechanisms for lymphocyte adhesion to cultured
endothelial cells. J Cell Biol 1988;107:321-331.
4. Oppenheimer-Marks N, Davis LS, Bogue DT, Ramberg J, Lipsky PE.
Differential utilization of ICAM-1 and VCAM-1 during the adhesion
and transendothelial migration of human T lymphocytes. J Immunol
1991;147:2913-2921.
5. Luscinskas FW, Cybulsky MI, Kieley JM, Peckins CS, Davis VM, Gimbrone MA Jr. Cytokine-activated human endothelial monolayers support enhanced neutrophil transmigration via a mechanism involving
both endothelial-leukocyte adhesion molecule-1 and intercellular adhesion molecule-1. J Immunol 1991;146:1617-1625.
6. Huang AJ, Manning JE, Bandak TM, Ratau MC, Hanser KR, Silverstein
SC. Endothelial cell cytosolic free calcium regulates neutrophil migration across monolayers of endothelial cells. J Cell Biol 1993;120:13711380.
7. Cooper D, Lindberg FP, Gamble JR, Brown EJ, Vadas MA. Transendothelial migration of neutrophils involves integrin-associated protein(CD47). Proc Natl Acad Sci U S A 1995;92:3978-3982.

hepa

WBS: Hepatology

HEPATOLOGY Vol. 26, No. 3, 1997

SAKAMOTO ET AL.

8. Volpes R, Van den Oord JJ, Desmet VJ. Immunohistochemical study ofadhesion molecules in liver inflammation. HEPATOLOGY 1990;12:59-65.
9. Burra P, Hubschher SG, Shaw J, Elias E, Adams DH. Is the intercellularadhesion molecule-1/leukocyte function associated antigen 1 pathwayof leukocyte adhesion involved in the tissue damage of alcoholic hepatitis? Gut 1992;33:268-271.
10. Mendenhall CL. Alcoholic hepatitis. In: Schiff L, Schiff ER, eds. Diseaseof the liver. Philadelphia: Lippincott, 1987:669.
11. Sato T, Tanaka J, Kono Y, Jones RT, Cowley RA, Trump BF. Hepatic
cellular injury following lethal Escherichia coli bacteria in rats. Lab
Invest 1982;47:304-310.
12. Komatsu Y, Shiratori Y, Kawase T, Hashimoto N, Han K, Shiina S,
Matsumura M, et al. Role of polymorphonuclear leukocytes in galactosamine hepatitis: mechanism of adherence to hepatic endothelial cells.
HEPATOLOGY 1993;20:1548-1556.
13. Lawrence MB, McIntire LV, Eskin SG. Effect of flow on polymorphonuclear leukocyte/endothelial cell adhesion. Blood 1987;70:1284-1290.
14. Lawrence MB, Smith CW, Eskin SG, McIntire LV. Effect of venous shear
stress on CD18-mediated neutrophil adhesion to cultured endothelium.
Blood 1990;75:227-237.
15. Peri G, Chiaffarino F, Bernasconi S, Martin-Padura I, Mantovani A.
Cytotoxicity of activated monocytes on endothelial cells. J Immunol
1990;144:1444-1448.
16. Weiss SJ. Tissue destruction by neutrophils. N Engl J Med 1989;320:
365-376.
17. Harlan JM, Schwartz BR, Reidy MA, Schwartz SM, Ochs HD, Harker L.
Activated neutrophils disrupt endothelial monolayer integrity by anoxygen radical-independent mechanism. Lab Invest 1985;52:141-150.
18. Itoh Y, Okanoue T, Enjo F, Sakamoto S, Takami S, Yasui K, Kagawa
K, et al. Regulation of hepatocyte albumin and a1-acid glycoprotein
secretion by monokines, dexamethasone, and nitric oxide synthase
pathway: significance of activated liver nonparenchymal cells. Dig Dis
Sci 1994;39:861-865.
19. Netland PA Zetter BR, Via DP, Voyta JC. In situ labelling of vascularendothelium with fluorescent acetylated low density lipoprotein. Histochem J 1985;17:1309-1320.
20. Tamatani T, Kotani M, Miyasaka M. Characterization of the rat leukocyte integrin, CD11/CD18, by the use of LFA-1 subunit-specific monoclonal antibodies. Eur J Immunol 1991;21:627-633.
21. Vollmar B, Menger MD, Glasz J, Leiderer R, Messmer K. Impact of
leukocyte-endothelial cell interaction in hepatic ischemia-reperfusion
injury. Am J Physiol 1994;267:G786-G793.
22. Inauen W, Granger DN, Meininger CJ, Schelling ME, Granger HJ, Kvietys PR. An in vitro model of ischemia-reperfusion–induced microvascular injury. Am J Physiol 1990;259:G134-G139.
23. Moldow CF, Jacob HS. Endothelial culture, neutrophil or enzymic generation of free radicals: in vitro models for the study of endothelial
injury. Methods Enzymol 1984;105:378-385.

AID

Hepa 0027/

5p26$$$521

08-13-97 09:26:48

663

24. Asako H, Kubes P, Wallance JL, Wolf RE, Granger DN. Modulation ofleukocyte adhesion in rat mesentric venules by aspirin and salicylate.
Gastroenterology 1992;103:146-152.
25. Jaeschke H, Farhood A, Smith CW. Neutrophil-induced liver cell injury
in endotoxin shock is a CD11b/CD18-dependent mechanism. Am J
Physiol 1991;261:G1051-G1056.
26. Issekutz AC, Lopes N. Endotoxin activation of endothelium for polymorphonuclear leukocyte transendothelial migration and modulation
by interferon-g. Immunology 1993;79:600-607.
27. Van OOsten M, Van de Bilt E, De Vries HE, Van Berkel TJC, Kuiper J.
Vascular adhesion molecule-1 and intercellular adhesion molecule-1
expression on rat liver cells after lipopolysaccharide administration in
vivo. HEPATOLOGY 1995;22:1538-1546.
28. Ohira H, Ueno T, Torimura T, Tanikawa K, Kasukawa R. Leukocyte
adhesion molecules in the liver and plasma cytokine levels in endotoxininduced rat liver injury. Scand J Gastroentorol 1995;30:1027-1035.
29. Weiss SJ, Young J, Lobuglio AF, Slivka A, Nimeh NF. Role of hydrogen
peroxide in neutrophil-mediated destruction of cultured endothelial
cells. J Clin Invest 1981;68:714-721.
30. Fujita H, Morita I, Murota S. A possible mechanism for vascular endothelial cell injury elicited by activated leukocytes: a significant involvement of adhesion molecules, CD11/CD18, and ICAM-1. Arch Biochem
Biophys 1994;309:62-69.
31. Siebeck M, Fink E, Weipert J, Jochum M, Fritz H, Spannagl M, Kroworsch P, et al. Inhibition of plasma kallikrein with aprotinin in porcine
endotoxin shock. J Trauma 1993;34:193-198.
32. Ogawa M, Mori Y, Ueda S, Mori T, Makino Y, Hori J, Ohto M, et al.
Protective effects of FUT-175 on acute massive hepatic necrosis inducedin mice following endotoxin injection and immunization with liver
proteins. J Hepatol 1993;9:393-400.
33. Shappell SB, Toman C, Anderson DC, Taylor AA, Entman ML, Smith
CW. Mac-1 (CD11b/CD18) mediates adherence-dependent hydrogen
peroxide production by human and canine neutrophils. J Immunol
1990;144:2702-2711.
34. Morita M, Watanabe Y, Akaike T. Inflammatory cytokines up-regulateintercellular adhesion molecule-1 expression in primary cultured mouse
hepatocytes and T-lymphocyte adhesion. HEPATOLOGY 1994;19:426431.
35. Essani NA, Fisher MA, Farhood A, Manning AM, Smith CW, Jaeschke
H. Cytokine-induced upregulation of hepatic intercellular adhesion
molecule-1 messenger RNA expression and its role in the pathogenesisof murine endotoxin shock and acute liver failure. HEPATOLOGY 1995;
21:1632-1639.
36. Ramond MJ. Poynard T, Rueff B, Mathurin P, Theodore C, Chaput JC,
Benhamou JP. A randomized trial of prednisolone in patients with severe
alcoholic hepatitis. N Engl J Med 1992;326:507-512.
37. Adams DH, Hubscher SG, Shaw J, Rothlein R, Neuberger JM. Intercellular adhesion molecule 1 on liver allografts during rejection. Lancet
1989;2:1122-1125.

hepa

WBS: Hepatology