C. Wayne Smith,1 and Hartmut. Jaeschket,**. *Department of Pathology,. University of Texas. Health. Science. Center,. Houston,. tDrug. Metabolism. Research.
Intercellular adhesion molecule 1 (ICAM-1) expression role in neutrophil-induced ischemia-reperfusion injury
and its in rat
liver Anwar Farhood,* Gerald M. McGuire,t Anthony C. Wayne Smith,1 and Hartmut Jaeschket,** *Department Biology
of Pathology,
Tokyo
Metropolitan
Department Medicine,
University
Inflammation
and
Institute
of Pediatrics, Houston
of Texas
Research,
The
of Medical and
* *
Health
Upjohn for
Science
Tokyo,
Experimental
to
ICAM-i mRNA levels increased during ischemia in the ischemic liver lobes; however, during reperfusion mRNA levels increased in both the ischemic and nonischemic lobes. Immunohistochemical evaluation indicated ICAM-i expression only on sinusoidal lining cells in controls; ischemia-reperfusion enhanced ICAM-1 expression in the sinusoids and induced some expression on hepatocytes The monoclonal anti-ICAM-i antibody iA29, but not an immunoglobulin G control antibody, administered at 1 h and 8 h of reperfusion (2 mg/kg) signifIcantly attenuated liver injury as indicated by 51% lower plasma alanine aminotransferase activities and 32-36% less hepatic necrosis at 24 h without affecting reactive oxygen formation by Kupffer cells and hepatic neutrophils. Although 1A29 reduced neutrophil extravasation in a glycogen peritonitis by 60%, the antibody had no signfficant effect on hepatic neutrophil infiltration during reperfusion. These data suggest that ICAM-1 plays a significant role during the neutrophil-dependent injury phase after hepatic ischemia and reperfusion and therefore blocking this adhesion molecule may have therapeutic potential against postischemic acute liver failure J. Leukoc. Biol. 57: 368-374; 1995.
.
integrins
Words: .
inflammation
Houston,
Kalamazoo,
Japan;
Speros
P
Therapeutics,
Miyasaka,S
Metabolism
Research
Department
Martel
Laboratory
Department
and
Cell
of Immunology, of Leukocyte
of Medicine,
The
Biology,
Baylor
College
of
oxygen
radicals
.
Kupifer
sion injury at a later stage (6-24 h of reperfusion) [7]. Because many neutrophils accumulate in the sinusoids, they can be involved in the pathogenesis by disturbing the microcirculation with a subsequent infarct [8, 9]. However, there is experimental evidence that neutrophils aggravate reperfusion injury by generating cytotoxic mediators such as reactive oxygen species [10]. In fact, functional inactivation of hepatic neutrophils, reducing their capability to generate reactive oxygen, had a substantial beneficial effect in this model [11]. The clearly documented involvement of neutrophils in the pathogenesis of reperfusion injury opens the possibility of inhibiting this inflammatory process, because the accumulation and the cytotoxicity of these cells are dependent on a variety of adhesion molecules not only on neutrophils but also on endothelial cells and parenchymal cells. Data demonstrated the importance of the /32 integrin Mac-i (CDllb/CD18) on neutrophils in hepatic ischemiareperfusion injury [11] as well as hepatic failure in endotoxin shock [12]. Because intercellular adhesion molecule 1 (ICAM-1) is a potential counterreceptor for Mac-i [i3] and both membrane proteins were shown to be important in transendothelial migration myocytes in vitro in the pathogenesis was investigated
[14] and in parenchymal cell injury of and in vivo [16-18], the role of ICAM-1 of hepatic ischemia-reperfusion injury evaluating tissue expression of ICAM-i
[15] by
and ICAM-1 mRNA in the beneficial effect of a monoclonal
liver
as well anti-ICAM-1
as
the potential antibody.
cells
peritonitis
INTRODUCTION Ischemia-reperfusion
AND
METHODS
Animals injury
of
the
liver
can
occur
during
hemorrhagic shock or hepatic surgery (trauma, tumor resection, transplantation). In experimental models it was recognized that reperfusion injury in the liver is mainly an inflammatory cell-mediated injury [1]. This injury is initiated by Kupifer cells generating increased amounts of reactive oxygen [2, 3]. Kupifer cells are activated during ischemia [4, 5] and are further stimulated by complement activation during reperfusion [6]. This initial reperfusion injury and complement activation cause infiltration of polymorphonuclear leukocytes (PMNs, neutrophils) into the postischemic liver [2]. Neutropenia experiments using a monoclonal antineutrophil antibody indicated that neutrophils contribute to reperfu-
Journal
tDrug
Michigan,-
MATERIALS
368
Masayuki
Texas
Abstract: The potential role of intercellular adhesion molecule-i (ICAM-1) in the pathogenesis of reperfusion injury was investigated in male Fischer rats subjected 45 mm of hepatic ischemia and 24 h of reperfusion.
Key
Center,
Company,
Sciences,
Center
M. Manning,t
of Leukocyte
Biology
Volume
57,
March
1995
Male from animals water.
Fischer rats (240-290 g body weight) were purchased Harlan Sprague Dawley (Indianapolis, IN). The had free access to food (Purina 5001 Lab Chow) and All animals received humane care in compliance with
Abbreviations: adhesion monoclonal leukocyte; Reprint 7256-300-210,
ALT,
molecule
1;
antibody; SOD,
alanine IgG, MPO,
The
dismutase;
Hartmut Upjohn
ICAM-l, G;
rnyeloperoxidase;
superoxide
requests:
aminotransferase;
immunoglobulin
PMN, TNF-a,
Jaeschke,
Company.
301
KC,
Street,
49001. Received
June
28,
1994;
accepted
November
necrosis
Metabolism
Henrietta 7,
cell;
mAb,
polymorphonuclear
tumor
Drug
intercellular
Kupffer
1994.
factor
a.
Research, Kalamazoo,
MI
the National Research Council’s criteria for the care and use of laboratory animals in research. All experimental protocols were approved by the animal protocol review committees of Baylor College of Medicine and The Upjohn Company.
deoxyribonuclease in Gey’s balanced salt solution, at room temperature for 20-30 mm. The digested tissues were strained through a layer of nylon mesh. The cell suspension was centrifuged at SOg for 2 mm to separate the parenchymal
Some of the animals were treated with the anti-ICAM-1 antibody 1A29 or an isotype-matched nonbinding immunoglobulin G (IgG) control antibody (clone SG 12/9). 1A29 binds to the ICAM-1 receptor on rat endothelial cells and blocks the function of the receptor [19]. Intravenous injection of 1A29 does not cause neutropenia. The clone for the antibody 1A29 was generated by M. Miyasaka [19] against purified rat ICAM-1 and the antibody was prepared as
cells from the nonparenchymal cells. Endothelial and different subpopulations of Kupifer cells (arbitrarily classified as KC1,2,3) were separated according to cell density by centrifugal elutriation at 875g using different flow rates. Endothelial cells were eluted at 23 ml/min flow rate. KC1, the smallest Kupifer cell fraction, were collected at 29 mI/mm flow rate. This cell fraction was not used in this study because it was
described previously body was generated limpet hemocyanin does not cross-react body (mAb) EU/mg).
[7]. The nonbinding control IgG antiby immunizing mice against keyhole as described [7]. The control antibody with rat tissue. The monoclonal anti-
preparations
lschemia-reperfusion
had
low
endotoxin
experiments
levels
(
z c
shown). To mRNA groups ischemic tative changes
support the immunohistochemical data, ICAM-1 levels were determined by Northern blot analysis in of four or five animals each of either controls or or postischemic livers. Northern blots of represenanimals are presented in Figure 2. To quantitate of ICAM-1 mRNA levels, the ratio between mRNA and the stably expressed mRNA of
ICAM-1
GAPDH was This approach
determined eliminates
to variation of levels increased
for these livers changes in RNA
RNA loading in the ischemic
on
the lobes
by densitometry. levels that are
activities
in both
groups
of animals
CTL I
I
5).
lh
II
However,
5h
II
ISCH
0.5-
Non-ISCH C) 0.0-
C
I
lh
5h
24h
Reperfusion
gel. ICAM-1 mRNA during ischemia and
(Fig.
1.0-
E
due Fig.
further increased during reperfusion (Fig. 3). After 5 and 24 h of reperfusion, mRNA levels declined; however, this effect may be due in part to the substantial injury in the postischemic lobes. In the nonischemic liver lobes, ICAM-1 mRNA increased only during reperfusion with the highest levels observed at 5 h of reperfusion (Fig. 3). To test the pathophysiological importance of ICAM-l expression in this model of inflammatory liver injury, the potential beneficial effect ofthe anti-ICAM-i mAb 1A29 was investigated. To evaluate the potency of our antibody preparation in vivo, its capacity to inhibit neutrophil accumulation in the peritoneum after glycogen administration was determined. Transendothelial migration is an essential step of neutrophil accumulation at the site of inflammation and requires Mac-i (CD11b/CD18)-ICAM-i interaction [13]. As shown in Figure 4, the antibody 1A29 reduced peritoneal neutrophil infiltration as indicated by a 60% lower myeloperoxidase activity. The results indicate that the antibody is able to inhibit ICAM-1-dependent transendothelial migration in vivo. Because of our previous documentation that neutrophils are not involved in the initial reperfusion injury [2] but only in the later phase [7], the first dose of the antibody was administered after 45 mm of ischemia and 1 h of reperfusion. The control group and the antibody-treated group had similar initial injury, as indicated by similar ALT
A
1.5-
a) -J
3.
Serniquantitative
ischeniic
(ISCH)
The
values
evaluation and
are
given
(I)
represents trol
the
.alu&s)
are
