SHOCK, Vol. 18, No. 2, pp. 152–157, 2002

INVOLVEMENT OF KUPFFER CELLS IN THE INTERACTION BETWEEN
NEUTROPHILS AND SINUSOIDAL ENDOTHELIAL CELLS IN RATS
Shinichi Sakamoto,* Takeshi Okanoue,* Yoshito Itoh,* Yoshio Nakagawa,*
Hideki Nakamura,* Atsuhiro Morita,* Yukiko Daimon,* Kyoko Sakamoto,†
Norimasa Yoshida,† Toshikazu Yoshikawa,† and Kei Kashima**Third Department of Internal Medicine and †First Department of Internal Medicine, Kyoto Prefectural
University of Medicine, Kyoto, Japan
Received 20 Jun 2001; first review completed 12 Jul 2001; accepted in final form 15 Oct 2001
ABSTRACT—During endotoxic liver injury, large numbers of neutrophils infiltrate the liver, and serum levels of tumor
necrosis factor-␣ (TNF-␣) become elevated. The object of this study was to assess the roles of TNF-␣ secreted by Kupffer
cells in the interaction between neutrophils and sinusoidal endothelial cells (SECs). Rat neutrophils were perfused onto
SECs that were stimulated with either TNF-␣ or supernatant from lipopolysaccharide (LPS)-stimulated Kupffer cells using
an in vitro flow system. Numbers of adhered or migrated neutrophils were counted, and the effect of an antibody againstintercellular adhesion molecule-1 (ICAM-1) was studied. Compared with controls (200 ± 21 cells/mm2), neutrophil adhesion to SECs was significantly increased by both TNF-␣ (342 ± 26 cells/mm2; P < 0.05) and LPS-stimulated Kupffer cellsupernatant (331 ± 29 cells/mm2; P < 0.05). Anti-ICAM-1 significantly inhibited neutrophil adhesion (139 ± 10 cells/mm2;
P < 0.05) and decreased the migration rate of neutrophils on SECs treated with LPS-stimulated Kupffer cell supernatant(P < 0.05). LPS-stimulated Kupffer cells secreted TNF-␣ in an LPS dose-dependent manner, and they significantly
enhanced ICAM-1 expression on SECs (P < 0.05 vs. control). In addition, dexamethasone suppressed TNF-␣ production
by LPS-stimulated Kupffer cells and decreased ICAM-1 expression and neutrophil adhesion on SECs. These findings
suggest that Kupffer cells are involved in neutrophil adhesion and migration in hepatic sinusoids via TNF-␣ production andinduction of ICAM-1 expression on SECs during liver injury associated with endotoxemia.
KEYWORDS—Endotoxin, liver injury, tumor necrosis factor-␣, intercellular adhesion molecule-1, dexamethasone

INTRODUCTION

Intercellular adhesion molecule-1 (ICAM-1), a member of theimmunoglobulin superfamily, is expressed on endothelial cells
and is upregulated by interleukin 1 (IL-1) and TNF-␣ (7). The
ligands of ICAM-1 are leukocyte integrins such as Mac-1(CD11b/CD18) on neutrophils (8) or lymphocyte functionassociated antigen 1 (LFA-1, CD11a/CD18) on lymphocytes(9). Modulation of adhesion molecules by monoclonal antibodies is reported to be useful in the inhibition of inflammatory
reactions and in preventing rejection of organ allografts (10).
Immunohistochemical studies have shown that several adhesion molecules are involved in the pathogenesis of various liverdiseases. Volpes et al. (11) observed that the expression of
ICAM-1 was increased on the sinusoidal endothelial cells(SECs) in inflamed lesions where LFA-1-positive lymphocytes
had infiltrated. Thus far, there have been few studies on theinteraction between neutrophils and SECs in vitro. We previously reported on the significant contribution of ICAM-1 andCD18 to neutrophil adhesion and migration in hepatic sinusoids using an in vitro flow system (12). This system allows forthe dynamic analysis of the spatial and temporal details ofcell-to-cell interactions under a programmed shear stress (13,14). In the present study, we employed this system to address
the hypothesis that TNF- ␣ , released by LPS-stimulated
Kupffer cells, plays a major role in the adhesion of neutrophils
to SECs in endotoxic liver injuries. We investigated thenumbers of adhered or migrated neutrophils to SECs that were
stimulated with either TNF-␣ or the supernatants of LPSstimulated Kupffer cells. Additionally, it has been reported
that corticosteroids improve neutrophil-mediated liver injury
induced by endotoxin (15). To assess the mechanism of theTumor necrosis factor-␣ (TNF-␣) is an inflammatory cytokine produced in response to endotoxin and is considered to bean essential mediator in the development of endotoxic shock(1). It has been shown that administration of endotoxin
increases serum levels of TNF-␣ (2). However, the mechanismof tissue injury due to endotoxic shock is still under investigation. In endotoxic liver injury, large numbers of neutrophils
accumulate in the liver tissue and are associated with the development of hepatocyte necrosis (2). Neutrophils play a majorrole in the pathogenesis of such disorders. This is supported bya previous report showing that the depletion of neutrophils
using anti-neutrophil antibody prevents endotoxin-induced
liver dysfunction in rats (3). On the other hand, Kupffer cells
contribute to septic liver injury by producing various chemical
mediators, including inflammatory cytokines (4). Administration of lipopolysaccharide (LPS) in rats stimulates the production of TNF-␣ by Kupffer cells and increases serum concentrations of TNF-␣ (5).
The involvement of leukocyte adhesion in the pathogenesisof the hepatic injury related to endotoxemia has clinicalrelevance. Leukocytes interact with the endothelial cells andextravasate prior to tissue infiltration. These phenomena are
mediated by specific adhesion molecules on these cells (6).

Address reprint requests to Takeshi Okanoue, MD, Third Department ofInternal Medicine, Kyoto Prefectural University of Medicine, KawaramachiHirokoji, Kamigyo-ku, Kyoto, 602-8655, Japan.
This study is supported in part by a grant-in-aid from the Ministry of Education,
Science and Culture of Japan (No. 07457139).

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anti-inflammatory effects of dexamethasone under these pathophysiological conditions, we investigated its effects on TNF-␣
production by LPS-stimulated Kupffer cells and ICAM-1
expression on SECs.
MATERIALS AND METHODS
Animals
Male Wistar rats (Shimizu Animal Inc., Kyoto, Japan; 200 to 250 g) were used.
They were fed a standard laboratory diet and water ad libitum under standard
laboratory conditions. Procedures were performed according to the guidelines of the
National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Isolation and culture of rat sinusoidal cells
Rat sinusoidal cells were obtained from male Wistar rats according to the method
previously described (16) with a slight modification. Briefly, the liver was perfused
via the portal vein with Ca++ and Mg++ free Hanks’ solution containing 0.01%
ethylenediaminetetraacetic acid and 10 U/mL of heparin. The flow rate was adjusted
to 22 mL/min. After 7 min, the liver was perfused with Hanks’ balanced salt
solution (HBSS) supplemented with 0.05% collagenase. After 5 min, the liver was
removed, cut into pieces, and passed through a mesh. Low-speed centrifugation(50g for 1 min) was then performed twice to separate hepatocytes and cell debris.
The suspension containing non-parenchymal cells was then introduced into acentrifugal elutriation rotor (Hitachi Koki Co., Ibaragi, Japan) to isolate the SECs
and Kupffer cells. Cells were loaded at a flow rate of 14 mL/min onto the elutriation
centrifuge head spinning at 2500 rpm at 4°C. To obtain SECs, the flow rate was kept
up to 20 mL/min, and 100 mL of cell suspension was collected. The flow rate wasthen adjusted to 26 mL/min, and 100 mL of suspension was collected to obtain
Kupffer cells. After centrifugation at 500g for 5 minutes at 4°C, the SECs were
resuspended at a density of 2 × 106 cells/mL in RPMI 1640 medium (Nissui
Pharmaceutical Co., Tokyo, Japan) containing 10% fetal calf serum (Invitrogen,
Carlsbad, CA), 10−7 M of dexamethasone (Sigma Chemical Co., St. Louis, MO),and 0.1% gentamycin sulfate (Sigma Chemical Co.). An aliquot (1.0 mL) of theendothelial cell suspension was incubated on type I C collagen-coated glass coverslips 24 × 50 mm (Matsunami Glass Industries, Osaka, Japan) for 2 h at 37°C in 5%
CO2. They were then rinsed and cultured in a medium free of dexamethasone for an
additional 10 h before the assay. To measure the expression of ICAM-1 by ELISA,
100 ␮L of the endothelial cell suspension (2 × 105 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 above.
Kupffer cell suspension was centrifuged at 500g for 5 min and was then resuspended at 1 × 106 cells/mL in the culture medium. This suspension (1.0 mL) was
incubated in a 24-well culture plate at 37°C in 5% CO2. After a 60-min incubation,
nonadherent cells were removed with a pipette. Sinusoidal cells were identified
morphologically by scanning electron microscopy, and histochemically using thefollowing cell markers: fluorescently labeled low-density lipoprotein (LDL) for
SECs (17) and anti-rat resident macrophage antibody (ED2; BMA Biochemicals,August, Switzerland) for Kupffer cells. About 92% of the cultured SEC fraction
revealed the characteristic features of pores and sieve plates, and took up acetylated
LDL. Flow cytometric analysis showed that ED2-positive cells exceeded 88% of theKupffer cell fraction.

Isolation of rat neutrophils
Rat neutrophils were obtained from the peritoneal fluid 4 h after an intraperitoneal injection of 15 mL of 0.1% glycogen (Sigma Chemical Co.). After washingwith HBSS, the cells were resuspended in HBSS at a density of 1 × 106 cells/mL andwere maintained at 4°C for 1 h. Giemsa staining showed >95% of the isolated cells
to be neutrophils. Flow cytometric analysis showed that 96% of the cell fraction
isolated from the peritoneal fluid expressed CD18 (WT.3; Seikagaku Corporation,
Tokyo, Japan) on their own surfaces (data not shown).

Supernatants of LPS-stimulated Kupffer cells and assay
of TNF-␣ production
After incubation for 1 h, cultured Kupffer cells were washed and stimulated with
serial concentrations of LPS (Escherichia coli, 055:B5, Difco Laboratories, Detroit,MI) with or without 10−7 M of dexamethasone. Supernatants were harvested after 1,
2, 6, and 12 h of incubation. To avoid the effects of residual LPS in the supernatantson the following assays, polymyxin B (Difco Laboratories), which neutralizes
biological function of LPS (18), was added to all of the supernatants after LPS
stimulation. They were stored at −30°C until the adhesion assay or the assay of
TNF-␣ production using an ELISA kit for rat TNF-␣ (Factor Test Rat TNF-␣ kit;
Genzyme Co., Cambridge, MA).

KUPFFER CELLS AND PMN ADHESION IN THE LIVER

153

Adhesion assay
The adhesive interaction between neutrophils and SECs was assessed using an invitro flow system where the fluid mechanics are well known, and surfaces can beeasily coated with cultured endothelial cells (13, 14). The chamber consisted of acollagen-coated cover slip with a confluent monolayer of SECs that was attached toa polycarbonate base. The space between the cover slip and the base was held
approximately 250 ␮m apart by a silastic rubber gasket. The flow rate wascontrolled with a syringe pump (Iwaki Glass Co.). Suspensions of neutrophils werepassed through the chamber at a wall shear stress of 0.5 dyn/cm2 and 37°C. Shearstress, ␶ in dyn/cm2, was calculated from the equation: ␶ ⳱ 3 ␮Q/2ba2, where ␶ is
the wall shear stress, ␮ is the viscosity of water at 37°C, which is used as an
approximation of the viscosity of HBSS (0.007 poise), Q is volumetric flow rate, ais the half channel height, and b is the channel width. The wall shear stress in thehepatic sinusoid was lower than that in the venule because of its slow blood flow.
A wall shear stress of 0.5 dyn/cm2 is supposed to be within pathophysiologicallimits in the hepatic sinusoids, based on the shear stress measured with intravitalfluorescence microscopy by Vollmar et al. (19). SECs on the coverslips were stimulated with 100 U/mL of rat TNF-␣ (Genzyme Co.) or the supernatants of LPSstimulated Kupffer cells for 4 h, mounted in flow chambers, and perfused for 1 minwith HBSS (rinse step), followed by an 8-min perfusion with a suspension ofneutrophils (perfusion step). The interaction between neutrophils and SECs wasobserved using phase-contrast microscopy (Olympus Co., Tokyo, Japan) and wasrecorded on a videotape. In some experiments, an antibody against rat TNF-␣ (100␮g/mL; Genzyme Co.) was added. The number of neutrophils that remained incontact with the endothelial cell monolayer was determined in six digitized framesof different fields of view from the last minute (7–8 min) of the perfusion step andwas expressed as the number of cells that adhered or migrated per square millimeter.
The neutrophils that adhered to the monolayer possessed spherical geometry andappeared as phase bright objects, whereas those that migrated beneath the monolayer were flat and appeared as phase dark objects, as previously described (14). The
migration rate was quantitated as follows:
the migration rate (%) ⳱ (migrated neutrophils × 100)/(adhered neutrophils+ migrated neutrophils)
In some experiments, the SECs were pretreated with anti-rat ICAM-1 monoclonal antibody (1A29; 5 ␮g/mL, Seikagaku Corporation) for 20 min at 37°C.

Expression of ICAM-1 on the SECs
The expression of ICAM-1 on the SECs was measured by cellular ELISA (3,12). SECs cultured on a 96-well plate coated with type I C collagen were incubatedwith 100 U/mL of rat TNF-␣ or the supernatants of Kupffer cells for 4 h and fixed
in 4% paraformaldehyde. In some experiments, an antibody against rat TNF-␣, or
10−7 M of dexamethasone was added. Three washes were done in PBS containing
0.1% bovine serum albumin (BSA). Nonspecific reactions were blocked by incubation for 30 min with 1% normal goat serum (Sigma Chemical Co.). After an
additional wash, the cells were incubated for 1 h with 50 ␮L of anti-rat ICAM-1
monoclonal antibody at a dilution of 1:1000. After three washes with PBS containing BSA, the cells were incubated for 1 h with a peroxidase-labeled antibody againstmouse IgG (Seikagaku Corporation). Peroxidase staining was then carried out witho-phenylenediamine (Sigma Chemical Co.) and H2O2. Absorbance at 490 nm wasmeasured by an Immunoreader (Multiskan; Labosystems, Helsinki, Finland). The
microplates were subsequently washed in tap water before 50 ␮l of 0.5% crystal
violet was added for 5 min. After a thorough wash in tap water, 100 ␮L of 33%
acetic acid was used to solubilize the nuclear stain, and absorbance at 570 nm wasmeasured, representing the actual cell count per well. The expression of ICAM-1
was determined as the ratio between the specific protein measurement and celldensity.

Statistical analysis
Values are expressed as means ± SE. Data were analyzed by analysis of varianceand Student’s t test, with Bonferroni’s correction for multiple comparisons. An
unpaired two-tailed Student’s t test was used to compare the migration rates. A levelof P < 0.05 was considered to be significant.

RESULTS
TNF-␣ production by LPS-stimulated Kupffer cells

The production of TNF-␣ from Kupffer cells stimulated with
serial concentrations of LPS for 6 h is shown in Figure 1A.
Although non-stimulated Kupffer cells showed no significant
TNF production, LPS stimulation induced a dose-dependent

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increase in the production of TNF-␣. Figure 1B shows the timecourse of TNF-␣ production by Kupffer cells stimulated with
100 ng/mL of LPS, which peaked at 6 h. Administration of
dexamethasone at a concentration of 10−7 M significantly
inhibited TNF-␣ production from LPS-stimulated Kupffer
cells.

SAKAMOTO ET AL.

TNF-␣ antibody attenuated this effect. The supernatants of
non-stimulated Kupffer cells had no effect on ICAM-1 expression. Dexamethasone attenuated the constitutive ICAM-1
expression on inactivated SECs and significantly suppressed
ICAM-1 expression on TNF-␣-stimulated SECs as well as on
SECs treated with the supernatant of LPS-stimulated Kupffer
cells.

Adhesion assay

Compared with controls (200 ± 21 cells/mm2), neutrophil
adhesion to SECs was significantly increased by both TNF-␣
(342 ± 26 cells/mm2; P < 0.05) and LPS-stimulated Kupffer
cell supernatant (331 ± 29 cells/mm2; P < 0.05; Fig. 2). These
were inhibited by the addition of neutralizing anti-TNF-␣ antibody (212 ± 28 cells/mm2; P < 0.05 and 249 ± 22 cells/mm2;
P < 0.05, respectively). Moreover, TNF-␣ and the supernatantof LPS-stimulated Kupffer cells significantly increased thenumber of neutrophils that migrated beneath the SEC. The
supernatants of non-stimulated Kupffer cells had no effect onneutrophil adhesion or migration. The addition of 5 ␮g/mL of
the anti-ICAM-1 significantly suppressed the increase in thenumber of neutrophils adhered to SECs stimulated with the
LPS-stimulated Kupffer cell supernatant (139 ± 10 cells/mm2;
P < 0.01; Fig. 2). In addition, the migration rate of neutrophils
through SECs treated with the supernatant of LPS-stimulated
Kupffer cells (21.2%) was significantly suppressed by theaddition of anti-ICAM-1 antibody (migration rate of 7.4%).
To investigate the effect of dexamethasone on Kupffer cellmediated neutrophil-SEC interaction, we examined neutrophil
adhesion and migration on SECs treated with the supernatantfrom LPS-stimulated Kupffer cells in the presence of 10−7 M
of dexamethasone. The numbers of both adhered and migrated
neutrophils (215 ± 20 and 28 ± 7 cells/mm2, respectively) weresignificantly decreased compared with those in the absence of
dexamethasone (P < 0.05).
Cellular ELISA for ICAM-1 expression on SECs

Stimulation with 100 U/mL of rat TNF-␣ significantly
enhanced the expression of ICAM-1 on SECs compared withunstimulated controls (Fig. 3). When the SECs were incubatedwith the supernatants of LPS-stimulated Kupffer cells, theexpression of ICAM-1 significantly increased. Anti-rat

DISCUSSION
The pathogenesis of endotoxin-induced liver injury is underintensive investigation using various animal models. In galactosamine/LPS-induced liver injury, numerous neutrophils infiltrate the liver, and necrosis of hepatocytes is demonstrated(20). Pretreatment with anti-neutrophil antiserum attenuates
the severity of the liver injury, indicating that neutrophils playa major role in this model (3). On the other hand, several
reports show that Kupffer cells contribute to endotoxic liver
injury by releasing several chemical mediators such as cytokines, proteases, and oxygen-derived free radicals (21, 22).
Gadolinium chloride, which destroys Kupffer cells by damaging their plasma membranes, decreases endotoxin-related liver
injury (23). Inflammatory cytokines including TNF-␣ havebeen implicated in this pathological condition. The administration of LPS induces the overproduction of TNF-␣ in a few
hours in rats in vivo (24). In the present study, we assessed therole of both purified TNF-␣ and TNF-␣ present in LPSstimulated Kupffer cell supernatants in neutrophil-SEC interactions.
We demonstrated that LPS induced a dose-dependent
increase in the production of TNF-␣ from Kupffer cells thatpeaked 6 h after stimulation. In support of this finding, Chensue et al. (25) had reported that positive staining for TNF-␣
was observed in Kupffer cells after LPS exposure in mice invivo. It has been suggested that TNF-␣ secreted from Kupffer
cells stimulates other sinusoidal lining cells, including SECs.
Our adhesion assay showed that the supernatant of LPSstimulated Kupffer cells, as well as TNF-␣, increased the adhesion of the neutrophils to the SEC monolayers under the shear
stress of hepatic sinusoids, and that anti-rat TNF-␣ antibody
suppressed neutrophil adhesion induced by LPS-stimulated

F IG . 1. TNF- ␣ production from Kupffer cells.
Values are expressed as mean ± SE in three experiments in triplicate. TNF-␣ levels increased LPS dose
dependently and peaked at 6 h (hr). Dexamethasone(DEX, 10−7 M) inhibited TNF-␣ production from LPSstimulated Kupffer cells. *P < 0.05 and **P < 0.01
compared with control. # P < 0.05 and ## P < 0.01
compared with DEX(-).

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KUPFFER CELLS AND PMN ADHESION IN THE LIVER

155

FIG. 2. Neutrophil adhesion to and migration
beneath SECs. Values are expressed as mean ± SE in
three experiments in duplicate. “%” shows migrated
neutrophil ratio to adhered neutrophil (migration rate).
TNF-␣ and the supernatant of LPS-stimulated Kupffer
cells [LPS (+) Kupffer cell] increased neutrophil adhesion
and migration, but the supernatant of
non-stimulated Kupffer cells [LPS (−) Kupffer cell] did
not. Dexamethasone (DEX) and anti–ICAM-1 antibody
decreased the number of both adhered and migrated
neutrophils. In addition, anti-ICAM-1 significantly inhibited the migration rate of neutrophils. *P < 0.05
compared with control. #P < 0.05 compared with TNF-␣
treated SECs. ##P < 0.01 compared with TNF-␣- treated
SECs.

Kupffer cells. Kupffer cells secrete various kinds of inflammatory cytokines other than TNF-␣ (25, 27). However, our finding using anti-TNF-␣ suggests that TNF-␣ secreted from
Kupffer cells, at least in part, promotes adhesion of neutrophils
in hepatic sinusoids, which is the first step in a sequential
cascade of inflammatory reactions. LPS-stimulated TNF-␣
production from our isolated Kupffer cells peaked at 6 h, but itpeaks at about 2 h in vivo (24). Knolle et al. (26) also reported
that isolated human Kupffer cells showed maximal secretion 6
h after LPS stimulation and that anti-inflammatory cytokine,
IL-10, suppressed TNF-␣ secretion from LPS-stimulated
Kupffer cells. The discrepancy of peak time between in vivo
and in vitro may be due to anti-inflammatory cytokines
secreted by other immune cells.
We previously reported that anti-ICAM-1 decreased migration rate of neutrophils on TNF-␣-stimulated SECs (12). In thisstudy, we showed that LPS-stimulated Kupffer cells increased
the number of neutrophils that migrated beneath the SEC
monolayers, and that anti-ICAM-1 decreased the migration
rate of neutrophils on SECs treated with the supernatants of
LPS-stimulated Kupffer cells. These results indicate that
ICAM-1 induced by LPS-stimulated Kupffer cells may be

crucial for neutrophil migration in hepatic sinusoids. Migrated
neutrophils produce oxygen-derived free radicals, which injure
hepatocytes (28). Inflammatory cytokines are also known to
induce the expression of ICAM-1 on hepatocytes as well as
SECs (29). Migrated neutrophils can recognize and cause
injury to hepatocytes overexpressing ICAM-1 (24). Our findings suggest that ICAM-1 on SECs plays an important role inthe migration step of neutrophils during the liver injury after
LPS exposure.
To assess the role of ICAM-1 in TNF-␣-induced neutrophilSEC interactions, we investigated ICAM-1 expression on
SECs after incubation with TNF-␣ or LPS-stimulated Kupffer
cells. Our cellular ELISA showed that the supernatant of LPSstimulated Kupffer cells, as well as 100 U/mL of TNF-␣,significantly increased ICAM-1 expression on SECs, and thatanti-rat TNF-␣ antibody led to the inhibition of ICAM-1
expression. Supernatants from non-stimulated Kupffer cells
had no effect on ICAM-1 expression. Additionally, the neutrophil adhesion assay revealed that anti-ICAM-1 antibody inhibited both adhesion and migration of neutrophils induced by
LPS-stimulated Kupffer cell supernatant. These findings
suggest that TNF-␣ released from LPS-stimulated Kupffer

FIG. 3. ICAM-1 expression on SECs. Values are themean ± SE of three experiments. TNF-␣ and LPSstimulated Kupffer cells increased ICAM-1 expression.
Dexamethasone significantly suppressed these effects.
*P < 0.05 compared with control. #P < 0.05 comparedwith TNF-␣-treated SECs. $P < 0.05 compared with
SECs treated with LPS-stimulated Kupffer cell supernatant.

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cells is crucial in the sinusoidal expression of adhesion
molecules and that ICAM-1 may play a major role in hepatic
infiltration of neutrophils in endotoxin-induced liver injury.
Shear stress is implicated in the interaction of leukocytes andendothelial cells mediated by adhesion molecules (13). Underconditions of physiological flow, firm adhesion of neutrophils
to endothelial cells cannot be elicited without rolling of neutrophils, which involves selectins (30). In fact, anti-ICAM-1
failed to inhibit the adhesion of neutrophils to IL-1-stimulated
endothelial cells at a shear stress of 4.0 dyn/cm2 (31).
However, at a shear stress below a physiological flow
(1.0 dyn/cm2), interactions between neutrophils and IL-1stimulated endothelial cells are induced by ICAM-1/CD18, but
do not involve the selectin-carbohydrate antigen (30). Shearstress in the hepatic sinusoids is very low because of its slow
blood flow (19). The contribution of ICAM-1 to hepatic infiltration of neutrophils in vivo is under controversy. Xu et al.
(32) reported that ICAM-1-deficient mice were resistant tolethal effects of high doses of LPS, and that this correlated witha significant decrease in neutrophil infiltration in the liver 24 hafter stimulation. This is consistent with our finding showingthat anti-ICAM-1 attenuated neutrophil adhesion to SECs
under low shear stress of hepatic sinusoids. On the other hand,
Jaeschke et al. (33) reported that anti-ICAM-1 inhibited liver
injury, but had no significant effect on neutrophil accumulationin hepatic sinusoids 90 min after endotoxin exposure. Vollmar
et al. (34) also stated that pretreatment with anti-ICAM-1 antibody was not effective in preventing intravascular sequestration of leukocytes in the liver 1 h after LPS administration.
However, the overexpression of ICAM-1 on SECs was notobserved until 6 to 8 h after LPS exposure (24, 35). This timeframe in vivo required to observe overexpression of ICAM-1
on SECs is consistent with our in vitro data showing that it
takes a few hours for Kupffer cells to generate TNF-␣ after
LPS-stimulation, and that ICAM-1 expression on SECs was
significantly increased at 4 h after treatment with TNF-␣.
Considering this temporal relationship, 1 h after LPS stimulation is supposed to be too early to estimate the role of ICAM-1
in the liver injury. In fact, normal and ICAM-1 deficient livers
had approximately similar numbers of neutrophils at 2 h,
whereas there were distinctly fewer neutrophils infiltrating theliver in ICAM-1-deficient mice 24 h after LPS exposure (32).
This finding shows that the roll of ICAM-1 in the late stage ofendotoxic liver injury differs from that in the early stage. In
this study, we clearly demonstrated that anti-ICAM-1 could
inhibit neutrophil adhesion to SECs with the overexpression of
ICAM-1, such as SECs in the late stage. Taken together, blocking ICAM-1-mediated cell interaction could be a therapeuticstrategy for preventing liver injury in endotoxic shock.
Corticosteroids have been used for the treatment in endotoxic liver injury, and may protect mice from LPS mortality inanimal models of endotoxic shock (36). In this study, we investigated the effect of dexamethasone on the neutrophil-SEC
interaction under a low-flow condition. Our adhesion assay
showed that dexamethasone significantly inhibited the increasein the numbers of the adhered and migrated neutrophils on
SECs treated with the supernatant of LPS-stimulated Kupffer
cells. This finding supports the anti-inflammatory effect of

SAKAMOTO ET AL.

dexamethasone. To clarify the mechanism that dexamethasone
decreases the neutrophil-SEC interaction, we examined theeffect of dexamethasone on TNF-␣ production from LPSstimulated Kupffer cells. Dexamethasone demonstrated asignificant suppressive effect on the production of TNF-␣ from
LPS-stimulated Kupffer cells. These findings were consistent
with an immunohistochemical analysis by Chensue et al. (25)showing that dexamethasone decreased Kupffer cell expressionof TNF-␣. We previously reported that TNF-␣ increases
ICAM-1 expression on SECs in a dose-dependent manner (12).
Thus, our findings suggest that the suppression of TNF-␣
release is one of the mechanisms that dexamethasone uses toattenuate ICAM-1-mediated inflammatory reactions in theliver.
On the other hand, our investigation demonstrated that dexamethasone inhibited neutrophil adhesion and ICAM-1 expression on SECs to an extent similar to that in control, althoughdexamethasone decreased TNF-␣ production from LPSstimulated Kupffer cells by only 50%. We also investigated theeffect of dexamethasone on ICAM-1 expression on SECs.
Cellular ELISA showed that dexamethasone (10−7 M) significantly decreased ICAM-1 expression not only on SECs treatedwith the supernatant of LPS-stimulated Kupffer cells, but also
on SECs stimulated with 100 IU/mL of TNF-␣. This findingmeans that dexamethasone can directly inhibit ICAM-1 expression on SECs in the presence of TNF-␣ secreted from Kupffer
cells. SECs have a constitutive expression of ICAM-1 in vivo
and in vitro (3, 12, 24). Our cellular ELISA demonstrated thatdexamethasone significantly attenuated the constitutive
expression on unstimulated SECs in vitro. Thus, we speculated
that 10−7 M dexamethasone could inhibit ICAM-1 expression
on SECs stimulated with Kupffer cell supernatants to the extent
similar to the control, whereas dexamethasone did notcompletely suppress TNF-␣ release. Dexamethasone might
prevent neutrophils from adhering to SECs mainly by inhibiting the overexpression of ICAM-1 induced by TNF-␣, in addition to the suppression of LPS-stimulated TNF-␣ release.
In summary, our studies suggest that TNF-␣ released from
LPS-stimulated Kupffer cells might induce the overexpressionof ICAM-1 on SECs in vivo, and ICAM-1, in turn, might playa major role in the hepatic infiltration of neutrophils in endotoxic liver injury.
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