www.elsevier.com/locate/ytaap

Toxicology and Applied Pharm

Role of osteopontin in hepatic neutrophil infiltration during

alcoholic steatohepatitisB

Udayan M. Apte, Atrayee Banerjee, Rachel McRee, Elizabeth Wellberg, Shashi K. Ramaiah*

Department of Pathobiology, College of Veterinary Medicine, Texas A&M University, MS4467 College Station, TX 77843-4467, USA

Received 23 June 2004; accepted 7 December 2004

Available online 10 May 2005

Abstract

Alcoholic liver disease (ALD) is a major complication of heavy alcohol (EtOH) drinking and is characterized by three progressive stages

of pathology: steatosis, steatohepatitis, and fibrosis/cirrhosis. Alcoholic steatosis (AS) is the initial stage of ALD and consists of fat

accumulation in the liver accompanied by minimal liver injury. AS is known to render the hepatocytes increasingly sensitive to toxicants such

as bacterial endotoxin (LPS). Alcoholic steatohepatitis (ASH), the second and rate-limiting step in the progression of ALD, is characterized

by hepatic fat accumulation, neutrophil infiltration, and neutrophil-mediated parenchymal injury. However, the pathogenesis of ASH is

poorly defined. It has been theorized that the pathogenesis of ASH involves interaction of increased circulating levels of LPS with

hepatocytes being rendered highly sensitive to LPS due to heavy EtOH consumption. We hypothesize that osteopontin (OPN), a matricellular

protein (MCP), plays an important role in the hepatic neutrophil recruitment due to its enhanced expression during the early phase of ALD

(AS and ASH). To study the role of OPN in the pathogenesis of ASH, we induced AS in male Sprague–Dawley rats by feeding EtOH-

containing Lieber–DeCarli liquid diet for 6 weeks. AS rats experienced extensive fat accumulation and minimal liver injury. Moderate

induction in OPN was observed in AS group. ASH was induced by feeding male Sprague–Dawley rats EtOH-containing Lieber–DeCarli

liquid diet for 6 weeks followed by LPS injection. The ASH rats had substantial neutrophil infiltration, coagulative oncotic necrosis, and

developed higher liver injury. Significant increases in the hepatic and circulating levels of OPN was observed in the ASH rats. Higher levels

of the active, thrombin-cleaved form of OPN in the liver in ASH group correlated remarkably with hepatic neutrophil infiltration. Finally,

correlative studies between OPN and hepatic neutrophil infiltration was corroborated in a simple rat peritoneal model where enhanced

peritoneal fluid neutrophil infiltration was noted in rats injected OPN intraperitoneally. Taken together these data indicate that OPN

expression induced during ASH may play a significant role in the pathogenesis of ASH by stimulating neutrophil transmigration.

D 2004 Elsevier Inc. All rights reserved.

Keywords: Alcohol; Lieber–DeCarli Diet; LPS; Osteopontin; Steatosis; Steatohepatitis

Introduction

Heavy alcohol (EtOH) consumption accounts for more

than 100,000 deaths per year in the US and public health

costs are more than $116 billion per year (NIAAA, 2001).

Alcoholic liver disease (ALD) is a major complication of

heavy EtOH consumption and is characterized by progres-

0041-008X/$ - see front matter D 2004 Elsevier Inc. All rights reserved.

doi:10.1016/j.taap.2004.12.018

i A portion of the data included in this paper were presented in the form

of a platform presentation at the 54th Annual Meeting of Association for

Study of Liver Diseases, Boston, MA, November 2003.

* Corresponding author. Fax: +1 979 845 9972.

E-mail address: sramaiah@cvm.tamu.edu (S.K. Ramaiah).

sive pathologic stages such as steatosis, steatohepatitis, and

cirrhosis (Diehl, 2002; Lieber and DeCarli, 1982; MacSween

and Burt, 1986; Maher, 2002; Nanji, 2002; Ramaiah et al.,

2004). Alcoholic steatosis (AS), is the initial stage of ALD,

characterized by extensive fat accumulation in the liver

along with mild to moderate liver injury (Galambos, 1972;

MacSween and Burt, 1986; Maher, 2002). Although

considered largely benign, recent investigations have

revealed that AS leaves the hepatocytes highly sensitive to

injury. The mechanisms behind such increased susceptibility

of steatotic liver include cellular changes due to fatty

metamorphosis (Bathgate and Simpson, 2002; Teli et al.,

1995), increased oxidative stress (Baykov et al., 2003;

acology 207 (2005) 25 – 38

Fig. 1. Experimental protocol to develop models of alcoholic steatosis and

steatohepatitis in rats. Male Sprague–Dawley rats (200–220 g) were fed

either a control or EtOH-containing Lieber–DeCarli diet for a period of 6

weeks to induce alcoholic steatosis. To generate a model of alcoholic

steatohepatitis, rats were fed either a control or EtOH-containing Lieber–

DeCarli diet for a period of 6 weeks, and then treated with a single dose

of LPS (10 mg/kg ip in saline) at the end of 6 weeks and sacrificed 24 h

later. Details of the experimental protocol have been described in

Materials and methods.

U.M. Apte et al. / Toxicology and Applied Pharmacology 207 (2005) 25–3826

Colell et al., 1998; Yang et al., 1997), decreased regener-

ative ability (Apte et al., 2004), and decreased expression of

peroxisome proliferators-activated receptors (Everett et al.,

2000; Fischer et al., 2003; Galli et al., 2001).

Alcoholic steatohepatitis (ASH), the second progressive

stage in ALD, is a rate-limiting step since a vast majority of

patients with ASH progress to cirrhosis even if they abstain

from drinking (Diehl, 2002; French, 2002; Nanji, 2002).

ASH is characterized by significant fat accumulation in the

liver (steatosis) in combination with neutrophil infiltration

(hepatitis), substantial liver injury, hepatic necrosis, and

apoptosis (Bautista, 2002; Diehl, 2002; Jaeschke, 2002;

Ramaiah et al., 2004). The mechanisms of pathogenesis of

ASH have been extensively studied and include increased

ROS/RNS production (Arteel, 2003; Lieber, 1990; Reinke

et al., 1987), nutritional deficit in the carbohydrates

(Korourian et al., 1999), enhanced pro-inflammatory cyto-

kines and chemokine levels (Hoek and Pastorino, 2002;

Wheeler et al., 2001), and high circulating levels of bacterial

endotoxin or lipopolysaccharide (LPS) in patients with ASH

(Arteel, 2003; Enomoto et al., 1999; Hoek and Pastorino,

2002; Uesugi et al., 2001). Irrespective of the mechanisms

of ASH, one of the most common pathological findings in

human ALD is the presence of neutrophils within the

hepatic parenchyma. However, the precise mechanism for

hepatic neutrophil infiltration and consequent hepatic injury

is not well understood. The role of cellular adhesion

molecules such as selectins, integrins (LFA-1/Mac-1), and

members of Ig gene superfamily (ICAM-1 and 2/VCAM-1)

has been demonstrated (Jaeschke, 2002; Jaeschke and

Smith, 1997a,b) as possible mechanisms for hepatic neutro-

phil infiltration.

In addition to adhesion molecules, recent evidence sug-

gests that a novel class of extracellular proteins called matri-

cellular proteins (MCP) play a critical role in the pathogenesis

of several inflammatory diseases (Francki and Sage, 2001;

Kim et al., 1997; Sodek et al., 2002).MCP are involved in cell

to cell and cell to matrix communication rather than structural

support unlike the ECM proteins. Osteopontin (OPN) is one

of the major MCP involved in cell to matrix communication

and in several inflammatory diseases including glomerular

nephritis (Cotran, 1999; Denhardt et al., 2001; Giachelli and

Steitz, 2000; O’Regan and Berman, 2000), inflammation

during CCl4-induced hepatotoxicity (Kawashima et al.,

1999), in puromycin-induced nephrotoxicity (Denhardt et

al., 2001), and nonalcoholic steatohepatitis (Sahai et al.,

2004). OPN is also a known chemoattractant for macro-

phages and neutrophils (Denhardt et al., 2001). However, the

role of OPN in hepatic neutrophil infiltration during ASH has

not been tested thus far. During ASH, neutrophils migrate to

the liver upon activation by cytokine and chemokine signals.

Once in the hepatic sinusoids they have to under go

transmigration to induce hepatocyte damage (Jaeschke,

2002; Jaeschke and Smith, 1997a,b; Jaeschke et al., 1991).

Transmigration involves movement of neutrophils from

hepatic sinusoids into space of Disse, which is the pseudo-

basement membrane in the liver. Neutrophils express specific

cell surface proteins such as h2 integrins [LFA-1 (CD11a/

CD18) and Mac-1 (CD11b/CD18)] and shed other cell

surface proteins such as L-selectin (CD62L) in response to

cytokine signals during neutrophil transmigration (Jaeschke

and Smith, 1997a,b). The primary objective of this study was

to investigate the role of OPN during steatohepatitis of ALD.

The hypothesized role of increased hepatic production of

OPN during early phase of ALD (AS and ASH) leading to

enhanced neutrophil infiltration in the liver, resulting in

hepatic injury was tested on this study. In the current study,

we examined the role of OPN in the development of ASH in a

rat model and examined the relationship between OPN and

neutrophil infiltration.

Materials and methods

Development of AS and ASH models. Simple models of AS

and ASH were developed based on the previous reports

(Deaciuc et al., 2002; Enomoto et al., 1999). Schematic

representation of the development of AS and ASH models is

shown in Fig. 1. Male Sprague–Dawley rats (220–250 g)

were purchased from Harlan Sprague–Dawley, Houston,

TX, USA, and were housed individually in cages in a

temperature-controlled animal facility with a 12-h light–dark

cycle. Rats were utilized after a 1-week equilibration period.

Development of AS model. Rats were divided into two

groups (n = 12 each), control diet group and experimental

diet group, and fed either control (isocaloric control diet

where the calories were adjusted with maltose-dextrin) or

EtOH-containing (35.5% of total calories) Lieber–DeCarli

diet (Bio-Serv, Frenchtown, NJ, #F1697SP) for a period of

U.M. Apte et al. / Toxicology and Applied Pharmacology 207 (2005) 25–38 27

6 weeks. For the first day, rats received plain liquid diet;

next, alcohol-treated rats received liquid diet containing

alcohol to 2% and 4% (w/v), each for 2 days. The 4% alcohol

diet was then continued for 6 weeks. The energy distribution

from the EtOH liquid is as follows: 18% protein, 35% fat,

and either 47% carbohydrate (control group) or 11.5%

carbohydrate (maltose dextrin) and 35.5% EtOH (EtOH-fed

groups). The food consumption was recorded daily and the

control rats were pair-fed according to the food consumption

of EtOH-fed rats. Rats were weighed at the beginning of the

study and weekly thereafter. Calories consumed by each rat

were measured daily. Rats were sacrificed at the end of

6 weeks (n = 4) by CO2 asphyxiation.

Development of ASH model. Following acclimatization,

rats were divided into four groups (n = 6 each group):

control, control + LPS, EtOH, and EtOH + LPS. EtOH and

EtOH + LPS group rats were fed Lieber–DeCarli liquid diet

(Bio-Serv, Frenchtown, NJ, #F1697SP) containing EtOH

(35.5% of total calories) for 6 weeks as described for the AS

model. Control and control + LPS group rats received an

isocaloric control diet where the calories were adjusted with

maltose–dextrin. At the end of 6 weeks, rats from control +

LPS and EtOH + LPS groups were injected with a single

injection of LPS (Escherichia coli 0111:B4, 10 mg/kg ip in

saline). The vehicle controls received an equal volume of

saline. All other rats were sacrificed at 24 h after LPS or

vehicle injection, which was given at the end of 6 weeks of

either control or EtOH containing Lieber–DeCarli diet

feeding. All animals were provided humane care in com-

pliance with the institutional guidelines (ULACC; Univer-

sity Laboratory Animal Care Committee) of Texas A&M

University.

Sample collection and processing. Blood was collected

from the dorsal aorta in heparinized tubes. Twenty micro-

liters of heparinized blood was separated in gas chromatog-

raphy vials (VWR, Bristol, CT) and submitted at an in-

house facility for estimation of blood alcohol content (BAC;

Department of Human Anatomy and Medical Neurobiology

at the Texas A&M Health Sciences Center, College Station,

TX). A fraction of heparinized plasma (approximately 0.5ml)

was employed for estimation of liver transaminase activities,

while the remaining plasma was snap frozen in liquid N2

and stored at �70 -C. Livers were harvested, weighed, and

divided into two parts. Three slices of the large and median

lobes were fixed in 10% neutral buffered formalin, while

remaining liver tissue was snap frozen in liquid N2 and

stored at �70 -C for further analysis.

Evaluation of liver injury. Liver injury was estimated by

plasma transaminase activities (alanine aminotransferase;

ALT and aspartate aminotransferase; AST) and corroborated

by histopathology of H&E-stained liver sections. Plasma

ALT and AST activities were analyzed on Vitros Chemistry

Analyzer (Ortho-Clinical Diagnostics, Raritan, NJ). For

histopathology, 4-AM-thick paraffin-embedded liver sec-

tions were cut and stained with H&E for bright-field

microscopy. Liver sections were evaluated for steatosis,

neutrophil inflammation, necrosis, and apoptosis. Hepatic

steatosis was scored using a system developed for the

intragastric infusion model of ALD as previously described

(Nanji et al., 1989) as follows: steatosis (the percentage of

hepatocytes containing fat) <25%, 1+; <50%, 2+; <75%,

3+; >75%, 4+. To assess the degree of inflammation, the

number of neutrophils per five high power fields was

quantitated from the H&E-stained liver sections. The

pathological alterations were evaluated and confirmed by

a board-certified pathologist.

Plasma endotoxin assay. Plasma endotoxin levels were

measured using a Limulus Amebocyte Lysate Endpoint

Assay Kit (BioWhittaker, Walkersville, MD). Pyrogen-free

procedures were employed throughout the assay. All plasma

samples were brought to room temperature prior to the

assay. The plasma samples were diluted (1:10) with LAL

Reagent water and heated in a 70 -C water bath for 5 min. A

pyrogen-free 96-well microplate was preheated at 37 -C.The standards and samples were then incubated with LAL

for 10 min at 37 -C followed by incubation with substrate

solution for 6 min. The reaction was stopped by adding 25%

acetic acid, and the absorbance was read at 410 nm using a

pyrogen-free microplate reader (Benchmark Plus, Bio-Rad,

Hercules, CA), preheated at 37 -C.

Assessment of apoptotic cells. Apoptotic cell death was

detected by morphological changes on light microscopy,

immunohistochemical localization of activated caspase-3

and by TUNEL assay. Apoptotic cells was visualized and

counted by TUNEL assay using ApopTag detection kit

(Serologicals Corporation, Norcross, GA) according to the

manufacturer’s protocol. Paraffinized liver sections of

samples from AS, ASH, and control groups were used.

Four slides per group per time point were stained and

apoptotic cells identified by dark brown nuclear staining

were counted under light microscope (Olympus BX45;

Olympus, Melville, NY). Apoptotic cells were identified by

dark brown nuclear staining and were quantified under light

microscope (Olympus BX51; Olympus, Melville, NY).

For immunohistochemistry of activated caspase-3, depar-

affinized 4 AM thick formalin-fixed paraffin-embedded

unstained liver sections were treated with 3% solution of

H2O2 in order to quench the endogenous peroxidase

activity. Sections were then incubated with blocking

solution (BioStain Rabbit IgG System, Biomeda, Foster

City, CA) to block nonspecific binding sites. An anti-

activated caspase-3 polyclonal antibody (Abcam, Cam-

bridge, MA, dilution of 1:100) was employed as a primary

antibody. Sections were then treated with a biotinylated anti-

rabbit secondary antibody followed by streptavidin (Bio-

Stain Rabbit IgG System, Biomeda, Foster City, CA). The

color was developed by exposing the peroxidase to

U.M. Apte et al. / Toxicology and Applied Pharmacology 207 (2005) 25–3828

diaminobenzidine reagent (Vector Laboratories, Burlin-

game, CA), which forms a brown reaction product. The

sections were then counterstained with Gill’s hematoxylin.

Activated caspase-3 expression was identified by the brown

colored cytoplasmic staining.

Three different approaches were employed to ensure

accurate detection of apoptosis: Firstly, the H&E-stained

sections were reviewed under light microscope for apoptotic

morphology such as condensation of chromatin and

pyknotic nuclei. Secondly, TUNEL staining was performed

and cells with definite nuclear dark brown staining were

quantified and compared with H&E sections. Thirdly,

activated caspase-3 immunohistochemical staining was

performed to visualize the cytoplasmic staining of apoptotic

cells. The results of these approaches were evaluated

simultaneously to quantify the degree of apoptosis in AS

and ASH.

RNA extraction and RT-PCR analysis of OPNmRNA. Total

RNA was extracted from frozen liver tissue after lysis and

homogenization using the RNeasy Midi kit (Quiagen,

Valencia, CA) according to the manufacturer’s protocol.

RNA purity was estimated by agarose gel electrophoresis

and spectrophometry. RT-PCR was carried out using

Titanium One-Step RT-PCR kit (BD Biosciences Clontech,

Palo Alto, CA). The primers employed in the RT-PCR

reaction were as follows: OPN forward-5VTCC AAG GAG

TAT AAG CAG AGG GCC A 3Vand reverse-5VCTC TTA

GGG TCT AGG ACT AGC TTG T-3V. The GAPDH RT-

PCR amplimer set (BD Bioscience Clontech, Palo Alto,

CA) was employed as an internal control. A total of 1 Ag of

total RNA was reverse transcribed to cDNA and further

subjected to PCR reaction at 94 -C for 45 s, 50 -C for 30 s,

and 72 -C for 1.5 min for 35 cycles. The PCR products (600

bp for GAPDH and 200 bp for OPN) were detected by 1%

agarose gel electrophoresis.

Western blot analysis for OPN. Liver cell lysates were

prepared in lysis buffer (1% Triton X-100, 50 mM NaCl, 10

mM Tris, 1 mM EDTA, 1 mM EGTA, 2 mM Na vanadate,

0.2 mM PMSF, 1 mM HEPES, 1 Ag/ml leupeptin, and 1 Ag/ml aprotinin) and protein concentration was estimated using

a Bio-Rad protein assay kit (Bio-Rad, Hercules, CA)

according to the manufacturer’s protocol. Briefly, 100 Agof cell lysate was resolved by electrophoresis on a 12%

sodium dodecyl sulfate (SDS) polyacrylamide gel (100 V,

1.5 h) in a running gel buffer containing 25 mM Tris, pH

8.3, 162 mM glycine, and 0.1% SDS. The samples were

transferred to nylon membrane for 3 h at 500 mA. The

membranes were incubated overnight in a cocktail contain-

ing rabbit polyclonal antibodies against recombinant human

OPN (LF-123 and LF-124, generous gift from Dr. Larry

Fisher, National Institute of Dental and Craniofacial

Research; 1:100 dilution in T-TBS with 0.1% Tween and

2% milk) overnight and subsequently probed with HRP-

conjugated anti-goat secondary antibody for 2 h at room

temperature. LF-123 recognizes the native (uncleaved) form

of OPN (¨68 kDa), while LF-124 recognizes the thrombin-

cleaved form of OPN (30 kDa; Rittling and Feng, 1998).

Final visualization was carried out with the enhanced

chemiluminescence kit (Pierce, Rockfor, IL). GAPDH was

used as an internal control to ensure equal loading of

proteins per well.

Immunohistochemistry. Hepatic OPN expression was

studied by immunohistochemical analysis conducted on

4-AM-thick formalin-fixed paraffin-embedded liver sec-

tions. Briefly deparaffinized 4-AM-thick paraffin-embedded

unstained liver sections were treated with 3% solution of

H2O2 in order to quench the endogenous peroxidase

activity. Antigen retrieval was achieved by citric acid

treatment. Sections were then incubated with 10% horse

serum in PBS to block nonspecific binding sites. An anti-

mouse OPN antibody (American Research Products, dilu-

tion of 1:500) was employed as a primary antibody. This

antibody recognizes both the native and cleaved forms of

OPN. Sections were then treated with a biotinylated rat-

adsorbed anti-mouse secondary antibody followed by

streptavidin (Vectastain Elite ABC Kit, Vector Laboratories,

Burligame, CA). The color was developed by exposing the

peroxidase to diaminobenzidine reagent (Vector Laborato-

ries, Burligame, CA), which forms a brown reaction

product. The sections were then counterstained with Gill’s

hematoxylin. OPN expression was identified by the brown-

colored cytoplasmic staining.

OPN ELISA. Serum levels of OPN in AS, ASH, and

control groups were determined using a commercially

available rat OPN ELISA kit (Assay Designs, Ann Arbor,

MI) according to the manufacturer’s protocol. The kit

utilizes polyclonal antibody to mouse OPN, immobilized

on a microtiter plate to bind the rat OPN in the standards and

sample. A rabbit polyclonal antibody to rat OPN labeled

with HRP is employed as a secondary antibody.

Culture and treatments of Hep G2 cells. To further test

the ability of EtOH and EtOH + LPS to induce OPN

expression and confirm the in vivo findings, 1 � 106 Hep

G2 cells were cultured in DMEM with 10% fetal calf

serum. Cells were then treated with increasing concen-

trations of EtOH (25, 50, 100, and 200 mM EtOH for

24 h). In a separate experiment, cells were treated with

either EtOH alone (100 mM) or EtOH + LPS (200 mM

and 1 Ag/Al, respectively) for 24 h. Following treatment

and incubation, cells were lysed in lysis buffer (1% Triton

X-100, 50 mM NaCl, 10 mM Tris, 1 mM EDTA, 1 mM

EGTA, 2 mM Na vanadate, 0.2 mM PMSF, 1 mM

HEPES, 1 Ag/ml leupeptin, and 1 Ag/ml aprotinin) on ice

and homogenates were prepared. Protein estimation was

carried out using Bio-Rad protein assay reagent (Bio-Rad,

Hercules, CA) and 100 Ag of protein was employed for

Western blotting of OPN as described before.

U.M. Apte et al. / Toxicology and Applied Pharmacology 207 (2005) 25–38 29

Cause and effect study to demonstrate the relationship

between OPN and neutrophil infiltration in an experimental

rat peritoneal model. Based on the correlative data

between hepatic neutrophil infiltration and OPN expression,

it is highly likely that OPN is most likely playing a role in

hepatic neutrophilic infiltration during ASH. However, to

confirm the correlative studies, an in vivo experiment was

designed to evaluate the chemotactic role of OPN in a rat

peritonitis model as described by Yao et al. (2003). Male

Sprague–Dawley rats (220–250 g) were allocated to 4

groups (n = 4 in each group). One group of 4 rats were

injected 5 mg of zymosan in 0.2 ml of PBS (pH 7.4) into the

subomental space which served as a positive control for

peritoneal neutrophilic infiltration. This protocol is known

to induce neutrophilic infiltration and peritonitis, 4 h post-

zymosan injection. The second group received a control

solution (0.2 ml of PBS). The third group received 100 Agof OPN in PBS (pH 7.4), and the fourth group received 100

Ag of OPN in thrombin (OPN incubated with thrombin for

30 min at 37 -C yielded cOPN). All solutions were

irradiated under UV to ensure sterility. Rats were then

sacrificed at 4 h post-treatment for evaluation of total WBC

and neutrophil response. Before sacrifice the rats were

injected intraperitoneally with 5 ml of ice-cold sterile PBS

containing 20 U/ml of heparin. The abdomen was massaged

for 2 min and then via a midline abdominal incision, free

fluid was recovered from the peritoneal cavity. This fluid

was pooled before being centrifuged at 1000g for 5 min at

4 -C to separate fluid and cellular components. Supernatants

were removed and evaluated for the numbers of leukocytes

using a Abbott Cell-dyn 3700 hematology analyzer (Abbott

Labs, Abbott Park, IL) and confirmed by Beckman T543

cell counter (Beckman Coulter Inc., Brea, CA). Cytospins of

the peritoneal cells were prepared by centrifugation at 500g

for 5 min and these cytospins were stained using Diff-Quik

and the differential counts were determined by light micro-

scopy. The total number of leukocytes and absolute

neutrophil numbers in the OPN-treated group were com-

pared with the zymosan-treated group and vehicle controls

to determine the chemotactic potential of OPN.

Statistics. Group comparisons were performed using

independent t test. A one-way analysis of variance was

used to determine statistical significance that might exist

between more than two distributions or sample groups.

Statistical analyses were made using SPSS 10.0 software

(SPSS Inc., Chicago, IL). Statistical significance was set at

P � 0.05.

Results

Simple models of alcoholic steatosis and steatohepatitis

EtOH alone administration for 6 weeks resulted in

significant fat accumulation (AS), while EtOH adminis-

tration for 6 weeks followed by LPS injection (10 mg/kg,

ip) resulted in fat accumulation along with neutrophil

infiltration, oncotic necrosis, and apoptosis (see below),

which are characteristic of human ASH. The pathology

noted in both AS and ASH is similar to that noted in

human cases of ALD (Diehl, 2002; MacSween and Burt,

1986; Maher, 2002; Nanji, 2002). Body weight gain of

rats in both AS and ASH group was not different from

the control diet-fed rats (333 T 7 in controls vs. 328 T 15

in EtOH-fed) indicating no nutritional differences between

the rats in AS, ASH, and control groups. The average

blood alcohol content in EtOH-fed rats was 83.65 T16.75 mg/dl.

Liver injury, steatosis, inflammation, oncotic necrosis, and

apoptosis

Liver injury estimated by plasma ALT (Fig. 2A) and

AST activities (Fig. 2B) indicated that rats in AS group

developed minimal to no liver damage. Following LPS

challenge plasma transaminase activities increased signifi-

cantly in the rats in ASH group indicating extensive

hepatocellular injury. The H&E-stained liver sections

corroborated the plasma transaminase activity data with

minimal to none hepatocyte necrosis in AS group (Fig. 3C)

and extensive multifocal neutrophil infiltration, and oncotic

coagulative necrosis in the ASH group (Fig. 3D). There was

no distinct zonality pattern in neutrophil infiltration and

necrosis present. The increase in AST observed in control

rats treated with LPS was minimal and histological sections

corroborated minimal necrosis in control and control + LPS-

treated groups (Figs. 3A and B). Scoring of H&E-stained

liver sections from AS group for steatosis (Fig. 4A) and

ASH group for inflammation (Fig. 4B) suggested extensive

steatosis in the rats with AS and significant neutrophil

inflammation in the ASH group. The control + LPS-treated

rats experienced moderate inflammation. There was a lack of

definite regiospecificity in the distribution of fat, neutrophil

infiltration, and coagulative necrosis in the EtOH + LPS-

treated rats. The histopathological observation of H&E-

stained liver sections corroborated the ALT and AST

activities.

Endotoxin levels

Endotoxin levels were detected in the plasma of control,

control + LPS, EtOH, and EtOH + LPS-treated rats using a

standard limulus amebocyte lysate endpoint assay (Fig. 5).

EtOH treatment alone did not increase circulating levels of

LPS. However, increased endotoxin levels were noted in

control and EtOH-fed rats treated with LPS. There were no

significant differences in the levels of plasma endotoxin

between control + LPS and EtOH + LPS-treated rats,

suggesting that the enhanced liver injury in EtOH + LPS-

treated rats is not due to higher LPS levels alone but may

due to the interaction of EtOH and LPS exposure.

Fig. 2. ALT (A) and AST (B) activities in plasma of rats fed either control

or EtOH-containing Lieber–DeCarli liquid diet for 6 weeks followed by a

single injection of LPS or vehicle as described in Materials and methods.

EtOH + LPS-treated rats exhibited significant increase in liver injury as

demonstrated by elevation in plasma ALT and AST activities. *Value

significantly different than all other groups; ! value significantly different

than respective control group. Data are expressed as mean T SE, P � 0.05.

U.M. Apte et al. / Toxicology and Applied Pharmacology 207 (2005) 25–3830

Apoptosis

Early and late apoptotic cell death was detected by

immunohistochemical staining of activated caspase-3 (Fig.

6, upper panels A–D) and TUNEL assay, respectively (Fig.

6, lower panel). Both TUNEL assay (Fig. 6, lower panel)

and activated caspase-3 staining (Fig. 6, upper panel C)

indicated apoptosis in the rats from AS group. LPS

treatment further increased apoptosis in both the control

and EtOH-treated (ASH) rats, but the number of apoptotic

cells was significantly higher in the ASH group (Fig. 6,

lower panel and upper panels B and D).

OPN mRNA and protein expression in steatosis

RT-PCR analysis of OPN indicated extremely low levels

of OPN mRNA expression in the livers of control rats.

However, EtOH treatment for 6 weeks resulted in enhanced

OPN mRNA expression in the livers (Fig. 7A). Correspond-

ing to themRNAdata, EtOH treatment for 6weeks resulted in

significant induction in OPN protein as observed in the livers

of AS group rats by Western blot analysis (Figs. 8A and B)

and immunohistochemistry (Fig. 9). The circulating levels of

OPN did not change after EtOH treatment (Fig. 10).

OPN mRNA and protein expression in steatohepatitis

Based on the functional role of OPN in post-ischemic

macrophage infiltration (Persy et al., 2003), and macrophage

migration into hepatic necrosis (Kawashima et al., 1999), we

hypothesized that OPN is over-expressed in liver to attract

neutrophils. Furthermore, based on the enhanced biologic

activity of processed form (thrombin-cleaved) of OPN, we

measured the cleaved form of OPN and correlated with

neutrophil infiltration. LPS treatment to control and EtOH-

fed rats induced OPN mRNA expression, although EtOH +

LPS rats had slightly higher OPN mRNA (Figs. 7A and B).

Similar induction in both hepatic (Figs. 8 and 9) and plasma

levels (Fig. 9) of OPN were observed. The hepatocytes

surrounding the inflammatory foci stained maximally for

OPN following LPS challenge (Fig. 9D). The OPN expres-

sion was predominantly localized to hepatocytes. Although,

the control diet-fed rats treated with LPS exhibited an

induction in OPN in the liver and plasma, the OPN expression

in the ASH group was maximum and significantly higher

than the control + LPS group. The thrombin-cleaved form of

OPN was higher in the ASH group as compared to control +

LPS and AS group. The extent of hepatic neutrophil

infiltration significantly correlated with the induction of

cleaved form of OPN expression in the liver (R2 = 0.86).

OPN expression in Hep G2 cells

To further test the ability of EtOH and EtOH + LPS to

induce OPN, Hep G2 cells were incubated with increasing

concentrations of EtOH. EtOH induced OPN at 50 mM

concentration, but higher concentrations (100 and 200 mM)

failed to induce further OPN expression (Fig. 11). Hep G2

cells incubated with EtOH + LPS resulted in further

induction of OPN than EtOH alone. These in vitro data

provide additional evidence that OPN is induced following

EtOH and EtOH + LPS exposure.

Cause and effect study to assess OPN-mediated neutrophil

response

An experimental rat zymosan-induced peritonitis model as

described by Yao et al. (2003) was modified to confirm the

correlative data between neutrophil infiltration and OPN

expression in the alcoholic steatohepatitis model. Zymosan

injection intraperitoneally is known to significantly enhance

peritoneal neutrophil accumulation resulting in peritonitis

which was confirmed in our studies (Fig. 12). Zymosan-

induced increases in the peritoneal fluid total WBC count

(>2-fold) and the absolute neutrophil numbers (>50-fold)

were compared with OPN-treated group (Fig. 12). The

increase in total WBC count was contributed mostly by

neutrophils and macrophages. Lymphocytes and mast cell

numbers were not altered between the groups. OPN-treated

group (both uncleaved and cleaved OPN) resulted in a

significant increase (>5000 cells in the uncleaved OPN group

Fig. 3. Representative photomicrographs of H&E sections of rats fed either control or EtOH-containing Lieber–DeCarli liquid diet for 6 weeks followed by a

single ip injection of LPS or vehicle as described in Materials and methods. EtOH feeding for 6 weeks resulted in significant steatosis, while injection of LPS

following EtOH led to steatohepatitis with neutrophil infiltration, oncotic necrosis, and apoptosis. (A) control; (B) control + LPS; (C) EtOH; (D) EtOH + LPS.

Magnification 20�. Block arrows indicate steatotic hepatocytes, arrowheads indicate neutrophil infiltration, and small arrows indicate necrotic hepatocytes.

Fig. 4. Steatosis score (A) and quantitation of neutrophils as an index of

inflammation (B) in the livers of rats fed either control or EtOH-containing

Lieber–DeCarli liquid diet for 6 weeks followed by a single ip injection of

LPS or vehicle. Steatosis was scored according to Nanji et al. as described

in Materials and methods. EtOH feeding resulted in significant steatosis

while LPS injection to EtOH-fed rats led to extensive neutrophil infiltration.

*Value significantly different than all other groups. Data are expressed as

mean T SE, P � 0.05.

U.M. Apte et al. / Toxicology and Applied Pharmacology 207 (2005) 25–38 31

and >2000 cells in the cleaved OPN group) in peritoneal total

WBC count and absolute neutrophil numbers (>1500 cells)

compared to the control group. In fact, the total WBC count

and absolute neutrophil number matched the zymosan-

treated group (Fig. 12). The cleaved-OPN-treated group did

not significantly differ in total WBC count and absolute

neutrophil count compared to the uncleaved-OPN group.

These results clearly suggest the involvement of OPN in

neutrophil infiltration and confirm the correlative findings of

OPN induction and neutrophil infiltration in the alcoholic

steatohepatitis model.

Fig. 5. Endotoxin levels in rats fed either control or EtOH-containing

Lieber–DeCarli liquid diet for 6 weeks followed by a single ip injection of

LPS or vehicle as described in Materials and methods. Endotoxin was

measured in the plasma of rats by limulus amebocyte lysate assay.

Fig. 6. Detection of apoptotic cell death by TUNEL assay and immunohistochemical staining of activated caspase-3 in the livers of rats fed either control or

EtOH-containing Lieber–DeCarli liquid diet for 6 weeks followed by a single injection of LPS or vehicle as described in Materials and methods.

Representative photomicrographs (upper panels A–D, magnification 40�) of activated caspase-3 staining and quantitation of apoptotic cells in liver sections

following TUNEL assay (lower panel). Arrows indicate positive activated caspase-3 hepatocyte staining. *Value significantly different than all other groups;! value significantly different than respective control group. Data are expressed as mean T SE, P � 0.05.

U.M. Apte et al. / Toxicology and Applied Pharmacology 207 (2005) 25–3832

Taken together, the correlative data and the cause and

effect studies strongly suggest that OPN expression is

induced in the AS and ASH. Furthermore, it appears that

expression of the cleaved form of OPN is enhanced following

LPS treatment with higher levels of cleaved form in the ASH

group.

Discussion

Our results demonstrate significant OPN induction

during initial stages of EtOH exposure and is further

enhanced following LPS administration. The correlation

between hepatic OPN induction and neutrophil infiltration

indicate that an increase in OPN creates a microenvironment

conducive for neutrophil inflammatory response following

heavy EtOH exposure and facilitates neutrophil transmigra-

tion into liver. Correlation studies between OPN and hepatic

neutrophil infiltration is corroborated by the preliminary

cause and effect studies.

The simplified experimental models of AS and ASH are

based on the previous investigations (de la Hall et al., 2001;

Enomoto et al., 1999; Murohisa et al., 2002; Tamai et al.,

2002) utilizing the interaction of chronic EtOH consumption

Fig. 7. OPN mRNA expression in rats fed either control or EtOH-

containing Lieber–DeCarli liquid diet for 6 weeks as followed by a single

ip injection of LPS or vehicle described in Materials and methods. OPN

mRNA detection by RT-PCR (A) and densitometric analysis (B). GAPDH

was employed as an internal control for RT-PCR to ensure equal loading

of RNA.

U.M. Apte et al. / Toxicology and Applied Pharmacology 207 (2005) 25–38 33

combined with a single LPS exposure. In our model, we

were able to demonstrate the complete spectrum of

pathology associated with ASH including steatosis, neu-

trophilic (mild mononuclear) inflammation, hepatocyte

oncotic necrosis, and apoptosis. These simple models of

alcoholic steatosis and steatohepatitis are highly reprodu-

cible, perfectly mimic the human ALD, and allows for

detailed mechanistic investigations into neutrophil trans-

migration and consequent liver injury.

The process of hepatic neutrophil infiltration and sub-

sequent liver injury involves endothelial and neutrophil

adhesion molecules (Shi et al., 1996). Adhesion molecules

such as intracellular adhesion molecules-1 (ICAM-1) and

vascular endothelial cell adhesion molecule-1 (VCAM-1)

present on hepatocytes and endothelial cell, respectively,

and LFA-1, Mac-1 (h2 integrins), and VLA-1 (h1 integrins)

on neutrophils play an important role in the extravasation

and cellular adhesion process (Bautista, 1997; Jaeschke,

2002; Jaeschke and Smith, 1997a,b; Jaeschke et al., 1991).

Nevertheless, it is clear that the cellular adhesion molecules

alone are not enough for neutrophil extravasation (Jaeschke,

2002). Based on our studies, it appears that OPN is an

important intra-hepatic signaling molecule in the develop-

ment of ASH (Fig. 13).

OPN is a secreted glycoprotein and has been well studied

in inflammatory diseases such as glomerular nephritis

(O’Regan and Berman, 2000), nonalcoholic steatohepatitis

(Sahai et al., 2004), cell-mediated immunity, and granuloma

formation (Giachelli and Steitz, 2000), ischemic injury, and

CCl4-mediated liver injury (Kawashima et al., 1999). OPN

has been shown as a chemotractant for macrophages and

neutrophils both in in vitro assays and in vivo experimental

conditions (Davis and Bayless, 2002). OPN has an RGD

domain that binds to avh3 integrins and mediates its cellular

effects (Bayless and Davis, 2001; Denhardt et al., 2001;

Smith et al., 1998). Furthermore, OPN is further cleaved by

thrombin in a smaller form (¨30 kDa), which has higher

chemotactic potential than the native form of OPN (Denhardt

et al., 2001; Giachelli et al., 1998). The thrombin-mediated

cleavage of OPN reveals a hidden domain on the proteins

(SVVYGLR domain), which possibly interacts with a9h1

integrin on neutrophils (Fig. 13). This interaction of cleaved

form of OPN with a9h1 integrin via SVVYGLR domain is

known to activate lymphocytes and neutrophils (Denhardt

et al., 2001; Giachelli et al., 1998; Yokosaki et al., 1999).

In our study, thrombin-cleaved form of OPN was induced

following EtOH exposure during AS and further induction

is observed after LPS administration in ASH (Fig. 8).

Although cleaved form of OPN correlated with neutrophil

infiltration, the expression of uncleaved native form of OPN

during steatosis is an interesting observation. The reason for

enhanced expression of OPN during AS, at a stage when no

neutrophil infiltration is observed can be attributed to the

lack of expression of the cleaved form of OPN. Thrombin-

cleaved OPN was higher in EtOH + LPS (ASH)-treated rats

as compared to EtOH (AS) alone-treated rats. One

possibility for the enhanced cleaved form of OPN and

consequent neutrophil transmigration during ASH can be

attributed to the higher post-translational modification of

OPN. Similar mechanisms may not be operating during

steatosis and thus may explain the lack of neutrophil

transmigration (Fig. 13). This notion is further supported

by the high correlation between higher thrombin-cleaved

form of OPN and neutrophil infiltration in the ASH group

(R2 = 0.86). This interpretation can be justified by the recent

finding that increased chemotaxis of monocytes by throm-

bin-cleaved form of OPN is demonstrated in murine models

of rheumatoid arthritis generated using LPS (Yamamoto et

al., 2003). The ability of EtOH and EtOH + LPS to induce

hepatic OPN expression was further corroborated in vitro

using HepG2 cells (Fig. 11). There is however a higher

baseline level of OPN expression which can be attributed to

the fact that HepG2 cells are of tumor cells in origin. A

recent report has shown that HepG2 cells constitutively

express OPN and constitutive OPN expression is implicated

in cancer development, progression and metastasis (Zhang

et al., 2004).

Being aware that correlation between OPN expression and

hepatic neutrophil infiltration does not necessarily mean a

causative relationship, an in vivo experiment was designed to

evaluate the chemotactic role of OPN in a model similar to

zymosan-induced rat peritonitis as described by Yao et al.

(2003). Using this model we have now demonstrated the

Fig. 9. Representative photomicrographs of immunohistochemical localization of OPN during alcoholic steatosis and alcoholic steatohepatitis. The arrows point

to the localization of OPNwithin the hepatocytes. Magnification 20�. (A) Control; (B) control + LPS; (C) EtOH (AS); and (D) EtOH + LPS (ASH). Brown color

indicates OPN-positive staining. (For interpretation of the references to colour in this figure legend the reader is referred to the web version of the article.)

Fig. 8. OPN protein expression in rats fed either control or EtOH-containing Lieber–DeCarli liquid diet for 6 weeks as followed by a single ip injection of LPS

or vehicle described in Materials and methods. OPN protein detection by Western blot (A) and densitometric analysis (B). GAPDH was employed as an internal

control for Western blot to ensure equal loading of RNA.

U.M. Apte et al. / Toxicology and Applied Pharmacology 207 (2005) 25–3834

Fig. 10. OPN levels in plasma of rats fed either control or EtOH-containing

Lieber–DeCarli liquid diet for 6 weeks followed by a single injection of

LPS or vehicle as described in Materials and methods detected by rat-

specific OPN ELISA kit (Assay Design, Ann Arbor, MI). *Value

significantly different than all other groups; !value significantly different

than respective control group. Data are expressed as mean T SE, P � 0.05.

Fig. 12. A comparison of total WBC count and absolute neutrophil numbers

in the peritoneal fluid of control, zymosan treated, uncleaved OPN (OPN),

and cleaved OPN (cOPN) group. Male SD rats were sacrificed at 4-h post-

treatment and peritoneal fluid was collected for WBC evaluation. *Value

significantly different from the control group. Data are expressed as mean T

SE, P � 0.05.

U.M. Apte et al. / Toxicology and Applied Pharmacology 207 (2005) 25–38 35

cause and effect relationship between OPN and neutrophil

infiltration. The OPN-mediated peritoneal neutrophil infiltra-

tion was comparable to the zymosan-induced neutrophilic

peritonitis group which served as a positive control. An

interesting observation in our zymosan-induced peritonitis

study is the higher peritoneal infiltration of monocytes/

macrophages in the OPN administered group. It appears that

OPN has higher affinity towards monocyte/macrophage

chemotaxis than neutrophils. This is not unexpected based

on the literature findings. A study from Kawashima et al.

(1999) have shown that osteopontin is known to act as a

chemokine that can induce hepatic monocyte migration

following carbon tetrachloride intoxication in rats. Although

there are high numbers of peritoneal neutrophil in the OPN-

treated group compared to controls, there is however an

interesting discrepancy between our correlative studies and

Fig. 11. Western blot analysis of OPN expression in Hep G2 cell following

24 h exposure to increasing doses of EtOH (panel A; 25, 50, 100, and 200

mM) and EtOH + LPS (panel B; 200 mM and 1 Ag/Al, respectively).

the cause and effect studies with regards to cleaved OPN and

neutrophil infiltration. Correlative studies in the alcoholic

steatohepatitis model showed high correlation between

higher thrombin cleaved form of OPN and neutrophil

infiltration. However, the cause and effect peritonitis model

did not show enhanced neutrophil numbers in the cleaved

OPN group compared to the uncleaved OPN group. This

discrepancy can be attributed to the insufficient amount of

cleaved OPN injected into the peritoneum. A dose–response

study with cleaved OPN and peritoneal neutrophil infiltration

should address this discrepancy and will be investigated in

our future studies. Clearly, based on the correlative and the

preliminary cause and effect studies, the role of OPN during

hepatic neutrophil infiltration is indicated. We are also

planning OPN knockout studies and OPN neutralizing

antibody intervention experiments to further confirm these

findings and clearly these studies should confirm the

definitive role of OPN in hepatic neutrophil infiltration.

Other possibilities for neutrophil transmigration during

ASH are worthy of investigation. In the present study we

have demonstrated that hepatic apoptosis occurs during AS

and ASH. This is similar to the reported literature findings

that the apoptotic cells appear to correlate well with the

occurrence of ASH (Casey et al., 2001; Deaciuc et al.,

2002). Lawson et al. (1998) have reported that excessive

parenchymal cell apoptosis may be an important signal for

transmigration of neutrophils. However, in the present study,

apoptosis may not be the primary signal for the following

reasons: (1) Although apoptosis was present in the AS and

ASH group, the apoptotic cells were not excessive enough

to overwhelm the macrophage scavenging system to result

in neutrophil migration. (2) The neutrophil migration in the

hepatic parenchyma appears to be around hepatocytes

undergoing oncotic coagulative necrosis and these necrotic

hepatocytes are sharply positive for OPN (Fig. 9). (3) EtOH

Fig. 13. The hypothesized role of OPN in hepatic neutrophil infiltration and features of OPN protein for neutrophil recruitment during steatosis and

steatohepatitis. EtOH and EtOH + LPS treatment result in induction of OPN in the liver. However, in the EtOH + LPS (ASH) group, the thrombin cleaved form

OPN is highly induced, correlating with significant neutrophil infiltration. Hepatic neutrophil infiltration is a consequence of neutrophil sequestration in the

portal vasculature, transmigration, and neutrophil adhesion to hepatocytes. The lack neutrophil infiltration in the EtOH alone group can be attributed to the

absence of thrombin cleavage.

U.M. Apte et al. / Toxicology and Applied Pharmacology 207 (2005) 25–3836

alone rats experienced significant apoptosis, however there

was a lack of neutrophil infiltration in that group. Together,

these data suggest that parenchymal cell apoptosis per se did

not result in neutrophil transmigration into the hepatic

parenchyma. Although, in our study the degree of apoptosis

correlated well with ASH, the apoptotic cells as a source of

hepatic OPN is unclear and needs additional investigation.

Other less likely mechanism for neutrophil transmigra-

tion is endotoxemia (Arteel, 2003; Wagner and Roth, 1999).

We noted only mild neutrophil infiltration in the LPS alone

group and marked infiltration in the EtOH + LPS group.

These findings suggest that the neutrophil infiltration is not

solely the result of endotoxemia but possibly a sequel to

EtOH + LPS combination. This is supported by the finding

that the OPN expression was highly induced in the EtOH +

LPS group compared to the LPS-alone group.

In addition to the OPN-mediated neutrophil transmigra-

tion mechanism, the mechanistic basis behind OPN induc-

tion is currently unknown. Based on the enhanced expression

of OPN following LPS challenge in EtOH-treated rats and in

HepG2 cells, it appears that LPS-induced cytokines such as

TNF-a (in vivo) and epidermal growth factor (HepG2 cells)

may be responsible for upregulation of OPN expression

(Pennington et al., 1998; Zhang et al., 2004), although not

tested in this study. In fact, recent studies have shown that

epidermal growth factor and pro-inflammatory cytokines can

upregulate OPN production (Sahai et al., 2004; Zhang et al.,

2004). There is no difference in the OPN mRNA between

LPS alone group and EtOH + LPS group. However, the

EtOH + LPS rats appear to have higher post-translational

cleavage of OPN based on the higher thrombin-cleaved form

of OPN resulting in a higher chemotactic form of OPN. It is

thus possible that LPS treatment results in increased

thrombin-mediated cleavage of OPN. Similar increased

chemotaxis of monocytes by thrombin-cleaved form of

OPN has been demonstrated in murine models of rheumatoid

arthritis generated using LPS (Yamamoto et al., 2003).

In summary, we have identified OPN (native and

thrombin-cleaved form) within the hepatocytes during ASH

rodent model and there appears to be a strong correlation

between cleaved form of OPN and hepatic infiltration. The

correlative findings are corroborated with the preliminary

cause and effect study demonstrated by higher peritoneal

neutrophil infiltration following OPN injection into the

peritoneal cavity. Based on our data, we propose a novel

mechanism for the pathogenesis of ASH (Fig. 13). The

mechanisms by which OPN signals to neutrophils remains to

be studied but based on the previous reports (Denhardt et al.,

2001; Giachelli et al., 1998), it can be speculated that OPN

directly interacts with neutrophils via a9h1 integrins.

Similarly, the role of native and thrombin-cleaved OPN in

neutrophil activation remains to be investigated.

U.M. Apte et al. / Toxicology and Applied Pharmacology 207 (2005) 25–38 37

Acknowledgments

Supported by the Center for Environmental and Rural

Health, NIH Seed Grant (NIEHS ES09106), and the Office

of Vice-President for Research Toxicology Seed Grant. The

authors wish to thank Catherine Samway, Clinical Pathol-

ogy technician, for her assistance with peritoneal fluid

analysis.

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