Download Liver cytokine production and ICAM

Am J Physiol Gastrointest Liver Physiol 292: G268 –G274, 2007.
First published September 7, 2006; doi:10.1152/ajpgi.00313.2006.
Liver cytokine production and ICAM-1 expression following bone
fracture, tissue trauma, and hemorrhage in middle-aged mice
Takeshi Matsutani,1,2 Shih-Ching Kang,1 Masao Miyashita,2 Koji Sasajima,2
Mashkoor A. Choudhry,1 Kirby I. Bland,1 and Irshad H. Chaudry1
1
Center for Surgical Research and Department of Surgery, University of Alabama, Birmingham, Alabama;
and 2Surgery for Organ Function and Biological Regulation, Department of Surgery I,
Graduate School of Medicine, Nippon Medical School, Tokyo, Japan
Submitted 14 July 2006; accepted in final form 12 August 2006
tumor necrosis factor-␣; interleukin-10; interleukin-6; hepatocellular
damage; intracellular adhesion molecule-1
are associated with the
development of systemic inflammatory response syndrome,
which may culminate in multiple organ dysfunction syndrome
(5, 6, 9, 12, 18, 20, 29, 34, 38, 39). Although systemic
inflammatory reactions by trauma-hemorrhage are characterized by elevated levels of inflammatory mediators, the exact
mechanisms by which trauma-hemorrhage leads to organ dysfunction have not been fully clarified. Furthermore, the depression in hepatocellular function following trauma-hemorrhage
persists despite fluid resuscitation (42). In this regard, the liver
is believed to play an important role in the occurrence of
multiple organ failure (42). Liver tissue has the highest population of macrophages in the body and therefore has great
MAJOR TRAUMA AND HEMORRHAGIC SHOCK
Address for reprint requests and other correspondence: I. H. Chaudry,
Center for Surgical Research, Univ. of Alabama, G 094 Volker Hall, 1670
Univ. Blvd., Birmingham, AL 35294-0019 (e-mail: Irshad.Chaudry@
ccc.uab.edu).
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potential for producing the local and/or systemic inflammatory
response (4, 5). Hepatocytes, Kupffer cells, and sinusoidal
endothelial cells participate in host-defense responses through
an orchestrated and complex intrahepatic network to respond to
various stress signals such as injury and infection (10). The
inflammatory responses after extrinsic stresses appear to result
from an unbalanced production of pro- and anti-inflammatory
mediators to maintain homeostasis. However, there are many
instances in which prolonged stress leads to the overproduction
of proinflammatory mediators, resulting in cellular, tissue, and
organ dysfunction.
Surgical and traumatic injuries influence the function of
leukocytes and endothelial cells, thus leading to various systemic responses (32). Cellular adhesion molecules are cell
surface glycoproteins that are critical for leukocyte adhesion to
the sinusoidal endothelium and transmigration and cytotoxicity
in a variety of inflammatory liver diseases (21, 30, 48).
ICAM-1 plays an important role in inflammation, and the
increased expression of ICAM-1 on endothelial cells is reflected in the activation of endothelial cells (30). ICAM-1 is of
particular importance since it mediates firm endothelial adhesion and facilitates leukocyte transmigration in the liver. Studies have shown that there is an upregulation of ICAM-1 on
both sinusoidal cells and hepatocytes in inflammatory liver
conditions such as hepatitis B viral infection, autoimmune liver
disorders, alcoholic hepatitis, and liver allograft rejection (1, 2,
7, 21, 37, 41, 48). It has also been reported that during
high-dose endotoxin shock, death is primarily due to liver
damage caused by the accumulation of neutrophils in liver
tissue (22, 24). Since the levels of ICAM-1 are elevated in the
bile and serum of patients with sepsis, measurement of circulating ICAM-1 facilitates the identification of those patients
with the highest risk of developing liver dysfunction (43).
These data thus support the hypothesis that adhesion molecules
are essential for producing inflammatory liver injury.
A potential limitation of all of the experimental studies has
been the exclusive use of animals that are 6 –12 wk of age. This
age range in mice corresponds approximately to an age of 4 – 8
yr old in humans (40). Furthermore, most of the previous
age-associated studies have been carried out using aged mice
(18 –24 mo), and no information is available concerning hepatocellular functions in middle-aged (12 mo old) mice following severe injury. Thus, although much has been learned about
the mechanisms of trauma-hemorrhage, this knowledge may
The costs of publication of this article were defrayed in part by the payment
of page charges. The article must therefore be hereby marked “advertisement”
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0193-1857/07 $8.00 Copyright © 2007 the American Physiological Society
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Matsutani T, Kang SC, Miyashita M, Sasajima K, Choudhry MA,
Bland KI, Chaudry IH. Liver cytokine production and ICAM-1 expression
following bone fracture, tissue trauma, and hemorrhage in middle-aged mice.
Am J Physiol Gastrointest Liver Physiol 292: G268–G274, 2007. First
published September 7, 2006; doi:10.1152/ajpgi.00313.2006.—Although
studies have indicated that hemorrhagic shock and resuscitation produces
hepatic damage by mechanisms involving adhesion molecules in endothelial
cells and hepatocytes, it is not known if there is any difference in the extent
of hepatic damage following bone fracture, soft tissue trauma, and hemorrhage (Fx-TH) between young and middle-aged animals. To study this,
young (6–8 wk) and middle-aged (⬃12 mo) C3H/HeN male mice were
subjected to a right lower leg fracture, soft tissue trauma, (i.e., midline
laparotomy), and hemorrhage (blood withdrawal to decrease the blood
pressure to 35 ⫾ 5 mmHg for 90 min) followed by resuscitation with four
times the shed blood volume in the form of lactated Ringer solution. Mice
were euthanized 24 h later, and liver tissues were harvested. Total bilirubin
levels in the hepatocyte extract increased markedly following Fx-TH in both
groups of mice; however, the increase in middle-aged mice was significantly
higher compared with young mice. TNF-␣ and IL-6 levels in the hepatocyte
extract following Fx-TH increased significantly in middle-aged mice but
remained unchanged in young mice. IL-10 levels significantly decreased in
middle-aged mice following Fx-TH but remained unchanged in young mice.
Kupffer cells from middle-aged mice produced significantly higher IL-6 and
IL-10 levels compared with young mice. Protein levels and mRNA expression of ICAM-1 in hepatocytes were also significantly higher in middle-aged
mice compared with young mice following Fx-TH. These results collectively
suggest that the extent of hepatic damage following Fx-TH is dependent on
the age of the subject.
EXPRESSION OF ICAM-1 IN THE LIVER FOLLOWING TRAUMA-HEMORRHAGE
MATERIALS AND METHODS
Animals. Male young (6 – 8 wk old) and middle-aged (⬃12 mo old)
C3H/HeN mice (Charles River Laboratories, Wilmington, MA) were
used in the experiments. Animals were housed with free access to
standard laboratory chow and water and maintained in a pathogen-free
animal facility for 2 wk before the experiments. Animal experiments
were carried out in accordance with the guidelines set forth in the
Animal Welfare Act and the National Institutes of Health Guide for
the Care and Use of Laboratory Animals and were approved by the
Institutional Animal Care and Use Committee of the University of
Alabama at Birmingham, AL.
Experimental groups. Operated animals were randomized into four
groups (with 6 – 8 mice/group): young mice with sham operation
(group 1), young mice with Fx-TH (group 2), middle-aged mice
with sham-operation (group 3), and middle-aged mice with Fx-TH
(group 4).
Fracture and trauma-hemorrhage procedure. Animals in the
Fx-TH groups were anesthetized with isoflurane (Minrad, Bethlehem,
PA) and restrained in the supine position. Mice were subjected to a
closed fracture of the right lower leg by three-point bending as
described previously (45). A 2.5-cm midline laparotomy (i.e., soft
tissue trauma) was performed and then closed aseptically in two layers
using 6-0 (Ethilon, Ethicon, Somerville, NJ) sutures. Subsequently,
both femoral arteries were aseptically catheterized with polyethylene-10 tubing (Clay-Adams, Parsippany, NJ) using a minimal dissection technique, and animals were allowed to awaken. Blood pressure
was monitored continuously through one of the femoral catheters
using a blood pressure analyzer (Digi-Med BPA-190, Micro-Med,
Louisville, KY). Upon awakening, animals were bled through the
other catheter to a mean arterial blood pressure (MAP) of 35 ⫾ 5
mmHg (prehemorrhage MAP was 90 ⫾ 5 mmHg), which was maintained for 90 min. At the end of that period, animals were resuscitated
with lactated Ringer solution (4 times the shed blood volume) over 30
min. Lidocaine hydrochloride was applied to the groin incision sites,
the catheters were removed, the vessels were ligated, and the incisions
were closed with 6-0 sutures. Sham-operated animals underwent the
same anesthetic and surgical procedures, but neither hemorrhage nor
fluid resuscitation was performed. After resuscitation, external fixation of the right lower leg by plastic tubing was performed. Buprenorphine (Reckitt & Colman, Richmond, VA) was injected subcutaneously immediately after surgery at a dosage of 0.1 mg/kg in all groups.
All procedures were operated aseptically, and there was no mortality
observed in this model of Fx-TH.
Preparation of hepatocytes and Kupffer cells. Animals were anesthetized by isoflurane 24 h after the operation. The circulating blood
AJP-Gastrointest Liver Physiol • VOL
was removed by a heart puncture. Hepatocytes and Kupffer cells were
isolated as described previously with some modifications (4). In brief,
the peritoneal cavity was opened aseptically, the portal vein was
exposed and catheterized with a 25-gauge winged infusion set, and the
vena cava was nicked. Retrograde perfusion of the liver was performed with 20 ml of HBSS (GIBCO-BRL, Grand Island, NY) at
37°C through the portal vein. This was immediately followed by a
perfusion with 20 ml of 0.05% collagenase type IV (162 U/mg, Sigma
Chemical, St. Louis, MO) in HBSS with 0.5 mM CaCl2 (Sigma
Chemical) at 37°C. The liver was then transferred to a petri dish
containing warm 0.05% collagenase, minced finely, incubated at 37°C
for 10 min, and passed through a sterile 150-mesh stainless steel
screen into a beaker containing 10 ml of cold HBSS and 10%
heat-inactivated FBS. Hepatocytes were then separated by low-speed
centrifuge at 50 g at 4°C for 3 min and washed in HBSS. The residual
cell suspension was then centrifuged twice at 400 g at 4°C for 15 min.
The supernatant was discarded, and the cell pellet was reconstituted
with 10 ml complete Williams’ E medium containing 10% heatinactivated FBS, 2 mM L-glutamine, and antibiotics of 50 U/ml
penicillin, 50 ␮g/ml streptomycin, and 20 ␮g/ml gentamicin (all from
GIBCO-BRL). The cell suspension was then layered over 16% metrizamide (Accurate Chemical & Scientific, Westbury, NY) in HBSS
and centrifuged at 2,300 g at 4°C for 45 min to separate Kupffer cells
from the remaining parenchymal cells in the pellet. Cells were washed
twice with centrifugation (800 g at 4°C for 10 min) with Williams’ E
medium and were incubated at 37°C with 5% CO2 and 95% humidity
for 2 h; nonadherent cells were removed, and the adherent Kupffer
cell population was gently removed and adjusted at a density of 1 ⫻
106 cells/ml in complete Williams’ E medium. Cells were then
cultured under the same conditions for another 24 h in the presence of
1 ␮g/ml LPS (from Escherichia coli, 0.55:B5, Sigma). After incubation, cell-free supernatants were obtained and stored at ⫺80°C until
assayed.
Hepatocyte preparation for cytokine determination. The hepatocyte
suspension was centrifuged twice at 50 g at 4°C for 3 min. The
supernatant was discarded, and the cell pellet was reconstituted with
homogenization buffer at 4°C; the buffer contained a protease inhibitor cocktail including (in mM) 10 HEPES (pH 7.9), 10 KCl, 1 EDTA,
0.5 EGTA, 1 DTT, 0.5 PMSF, 1 sodium fluoride, 20 ␤-glycerophosphate, and 1 sodium vanadate in PBS (pH 7.2) containing 0.5% Triton
X-100. Samples were sonicated for 1 min and incubated at 4°C for
1 h. The final homogenate was centrifuged at 120,000 g. The supernatants were used for the cytokine assay. Tissue protein content was
determined by a colorimetric assay using the Bio-Rad DC Protein
Assay (Bio-Rad, Hercules, CA).
Analysis of bilirubin, TNF-␣, IL-6, and IL-10 in hepatocyte extracts. The total bilirubin in hepatocyte extracts was measured as a
marker for cholestasis using a total bilirubin kit from Sigma Chemical.
TNF-␣, IL-6, and IL-10 were measured in hepatocyte extracts using a
sandwich ELISA technique as previously described by our laboratory
(26). TNF-␣ levels were determined by the DuoSet ELISA system
(R&D Research, Minneapolis, MN). IL-6 and IL-10 levels were
determined by the OptEIA system (BD Bioscience, San Diego, CA).
Expression of mouse ICAM-1 mRNA in hepatocytes. Total RNA
was extracted from hepatocytes using TRIzol (Life Technologies,
Gaithersburg, MD) according to the manufacturer’s instructions.
Briefly, hepatocytes were homogenized in a denaturing solution, and
RNA was sequentially isolated using phenol-chloroform extraction
and isopropanol precipitation. To prepare cDNA, 1 ␮g of total RNA
was reverse transcribed using murine Moloney leukemia virus reverse
transcriptase (Clontech) primed with random hexamers in a final
volume of 100 ␮l. PCR was performed using HotStarTag DNA
polymerase and the Q-Solution Kit (Qiagen, Valenicia, CA) in accordance with the manufacturer’s protocol. The primers used were
5⬘-TTCCGCTACCATCACCGTGTATTC-3⬘ (forward) and 5⬘-CTGGCCTCGGAGACATTAGAGAAC-3⬘ (reverse). Briefly, the PCR
assay was performed using 5 ␮l of the cDNA mixture. RT products
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have limited relevance to the adult patient population. Trauma,
critical care physicians, and general surgeons have long noted
anecdotal experience of a significant divergence in the response of the pediatric and adult populations to severe trauma.
The age-associated experiences were recently confirmed in a
retrospective analysis that showed the importance of sex and of
age in the outcomes of patients following traumatic injury (16,
17). In view of this, we examined whether young (6 – 8 wk of
age) and middle-aged (⬃12 mo of age) mice differed in the
immune and physiologic responses following bone fracture,
soft-tissue trauma, and hemorrhage (Fx-TH). The aim of this
study, therefore, was to determine whether middle age has any
influence on the extent of hepatic damage following Fx-TH.
The degree of hepatic damage was assessed by measurements
of bilirubin, TNF-␣, IL-6, and IL-10 in hepatocyte extracts and
ICAM-1 expression in hepatocytes following Fx-TH. The
ability of Kupffer cells to produce TNF-␣, IL-6, and IL-10 was
also measured.
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EXPRESSION OF ICAM-1 IN THE LIVER FOLLOWING TRAUMA-HEMORRHAGE
RESULTS
Total bilirubin levels. We directly compared the responses to
Fx-TH in young mice (6 – 8 wk of age) with those of middleaged mice (⬃12 mo of age) to determine the manner in which
these two age populations responded to the same injury. Based
on the life tables, 6- to 8-wk-old mice correspond to 4- to
8-yr-old humans and 12-mo-old mice correspond to 40-yr-old
humans (40).
To determine if there were any age-associated differences in
the response to Fx-TH, we assessed hepatocellular damage in
young and middle-aged mice at 24 h after Fx-TH. The degree
of hepatocellular damage was measured by bilirubin levels in
hepatocyte extracts (Fig. 1). Total bilirubin levels of young and
middle-aged mice after Fx-TH increased significantly compared with sham-operated mice in both age groups; however,
total bilirubin levels in middle-aged mice following Fx-TH
were significantly higher (⬃1.5- to 2-fold) than the levels in
young mice. There were no differences in bilirubin levels in
young and middle-aged sham-operated mice. These findings
show that hepatocellular damage occurred in our Fx-TH model
and that the damage was substantially increased in middle-aged
mice.
Inflammatory mediators in hepatocyte extracts. The levels of
TNF-␣ (A), IL-6 (B), and IL-10 (C) in the hepatocyte extract of
young and middle-aged mice at 24 h after Fx-TH are shown in
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Fig. 1. Levels of total bilirubin in hepatocyte extracts of young and middleaged C3H/HeN mice 24 h after bone fracture, soft tissue trauma, and hemorrhage (Fx-TH). Values are mean ⫾ SE; n ⫽ 6 – 8 mice/group. *P ⬍ 0.05,
Fx-TH vs. sham animals; #P ⬍ 0.05, young vs. middle-aged mice (by ANOVA
and Fisher’s post hoc test).
Fig. 2. TNF-␣ (Fig. 2A) and IL-6 (Fig. 2B) levels in young
mice did not alter following Fx-TH, whereas each of these
levels in middle-aged mice following Fx-TH increased significantly compared with Fx-TH in young mice and sham-operated mice in both age groups. IL-10 (Fig. 2C) levels in young
mice following Fx-TH were not different compared with IL-10
levels in sham-operated mice. IL-10 levels decreased in middle-aged mice following Fx-TH; however, the decrease in
IL-10 was not evident in young mice. These results clearly
demonstrate that there is an age-associated difference in the
inflammatory and anti-inflammatory cytokines present in the
hepatocyte extract following Fx-TH.
Kupffer cell cytokine production. As shown in Fig. 3, the
production of TNF-␣ (A), IL-6 (B), and IL-10 (C) by Kupffer
cells in young and middle-aged mice increased significantly at
24 h following Fx-TH. There were no significant differences in
TNF-␣ production between young and middle-aged mice. In
contrast, the production of IL-6 and IL-10 by Kupffer cells was
significantly higher in middle-aged mice compared with young
mice.
ICAM-1 mRNA by RT-PCR. To examine whether Fx-TH
induces ICAM-1 mRNA in the hepatocyte, we examined
mRNA levels of ICAM-1 in young and middle-aged mice 24 h
after Fx-TH (Fig. 4). Fx-TH did not produce any change in
ICAM-1 mRNA levels in young mice compared with shamoperated mice. ICAM-1 mRNA levels in middle-aged mice
were significantly higher compared with those in sham-operarted mice 24 h after Fx-TH. No significant differences in
Fx-TH-mediated increases were observed in young and middle-aged mice. There were no differences in these levels in
sham-operated mice in both groups. These results suggest that
Fx-TH induces ICAM-1 mRNA in hepatocytes in middle-aged
but not young mice.
ICAM-1 protein levels in hepatocytes. We examined
ICAM-1 protein levels in hepatocyte extracts by Western blot
assay following Fx-TH. Western blot analysis of hepatocyte
extracts from young and middle-aged mice in sham control
groups and Fx-TH groups are shown in Fig. 5. Young and
middle-aged mice showed the presence of ICAM-1 protein in
hepatocytes. Consistent with mRNA levels, there was a significant increase in ICAM-1 levels in hepatocytes of middle-age
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were used in a total volume of 50 ␮l containing 5 ␮l of 10⫻ PCR
buffer, 10 ␮l of Q-solution, 0.2 mM of each dNTP, 1.5 units of
HotStarTag polymerase (Qiagen), and 1 ␮M of each of the primers.
PCRs were carried out in a gradient Mastercycler (Eppendorf, Westbury, NY). For each PCR, preheating was at 95°C for 15 min, and
then annealing at 94°C for 40 s, 55°C for 45 s, and 75°C for 1 min,
with a final extension phase at 72°C for 10 min. Each reaction was
analyzed for amplification between 25 and 40 cycles. The amplification of ␤-actin (Clontech) was utilized as the housekeeping gene. PCR
products were analyzed by electrophoresis on 1.5% agarose gels in
1⫻ Tris-borate-EDTA buffer. The intensity of the bands was measured in a 500 Fluoresence ChemiImager (Alpha Diagnostics, San
Leandro, CA). The relative absorbance of the PCR products was
corrected against the absorbance obtained for ␤-actin.
Analysis of ICAM-1 in hepatocytes. The presence of ICAM-1 in
hepatocytes was assessed by Western blot analysis. Briefly, hepatocytes were homogenized, the volume was adjusted to 1 ml with 1⫻
SDS sample buffer [62.5 mM Tris 䡠 HCl (pH 6.8) at 25°C, 2% (wt/vol)
SDS, 10% glycerol, 50 mM DTT, and 0.01% (wt/vol) bromophenol
blue], and samples were boiled at 95°C for 5 min. An aliquot of the
extract (20 ␮l) was loaded on a Nu-PAGE 4 –12% bis-Tris gel and
electrophoresed at 200 V. Proteins were electroblotted to a nitrocellulose membrane and blocked with 5% BSA in 10 mM Tris 䡠 HCl (pH
7.6) buffer containing 150 mM NaCl and 0.1% Tween 20 for 1 h at
room temperature. The membrane was first incubated with goat
anti-ICAM-1 polyclonal antibody (Santa Cruz Biotechnology, Santa
Cruz, CA) diluted to 1:1,000 with the same buffer for 2 h and later
with the secondary antibody conjugated to horseradish peroxidase
(Santa Cruz Biotechnology) diluted to 1:3,000 with 10 mM Tris 䡠 HCl
buffer (pH 7.6) containing 150 mM NaCl, 0.1% Tween 20, and 5%
nonfat dry milk. After blots had been washed three times, immunoreactive bands were visualized by chemiluminescence using Chemiglow west substrate (Alpha Diagnostics).
Statistics. Data are presented as means ⫾ SE of 6 – 8 animals/
group. One-way ANOVA for multiple comparison followed by
Tukey’s or Dunn’s tests was employed to determine the significance
of the differences between experimental means. P ⬍ 0.05 was considered significant for all statistical analysis.
EXPRESSION OF ICAM-1 IN THE LIVER FOLLOWING TRAUMA-HEMORRHAGE
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Fig. 2. Levels of TNF-␣ (A), IL-6 (B), and IL-10 (C) in hepatocyte extracts of
young and middle-aged C3H/HeN mice 24 h after Fx-TH. Values are means ⫾
SE; n ⫽ 6 – 8 mice/group. *P ⬍ 0.05, Fx-TH vs. sham animals; #P ⬍ 0.05,
young vs. middle-aged mice (by ANOVA and Fisher’s post hoc test).
mice subjected to Fx-TH compared with the levels observed in
sham-operated mice. No significant differences were observed
in ICAM-1 levels in young mice subjected to Fx-TH or sham
operation.
DISCUSSION
The present study indicates that Fx-TH caused a substantial
increase in the bilirubin levels in hepatocyte extracts, indicating hepatocellular damage. Our results also indicate that ageassociated differences exist between tissue cytokine levels in
hepatocyte extract and cytokine production from LPS-stimulated Kupffer cells. Moreover, these cytokine responses following Fx-TH were significantly influenced by age and contributed to the increased hepatocellular damage in middle-aged
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Fig. 3. Production levels of TNF-␣ (A), IL-6 (B), and IL-10 (C) from Kupffer
cells with LPS stimulation. Twenty-four hours after Fx-TH in young and
middle-aged C3H/HeN mice, livers were aseptically removed and processed
for Kupffer cell isolation. Kupffer cells were cultured with 1 ␮g/ml LPS for
24 h, and supernatants were harvested for cytokine measurements. Values are
means ⫾ SE; n ⫽ 6 – 8 mice/group. *P ⬍ 0.05, Fx-TH vs. sham animals; #P ⬍
0.05, young vs. middle-aged mice (by ANOVA and Fisher’s post hoc test).
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mice compared with young mice. The major new finding of our
study is the observation that hepatocellular damage following
Fx-TH was associated with increased expression of ICAM-1 in
hepatocyte. In this regard, mRNA levels of ICAM-1 in middleaged mice were significantly higher than those in young mice.
Moreover, Western blot analysis demonstrated that the hepatocellular damage following Fx-TH in middle-aged mice was
associated with increased ICAM-1 expression.
Liver tissue has the highest population of macrophages in
the body and therefore has great potential for the production of
local and/or systemic inflammatory responses. It is well known
that trauma-hemorrhage activates Kupffer cells, and these cells
release proinflammatory cytokines, which may increase liver
damage by promoting neutrophil infiltration (4, 5). It is also
known that trauma-hemorrhage decreases the cytotoxic capacity of splenic and peritoneal macrophages, but it increases the
cytotoxic capacity of Kupffer cells. Previous studies from our
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EXPRESSION OF ICAM-1 IN THE LIVER FOLLOWING TRAUMA-HEMORRHAGE
laboratory (4, 5, 28) have demonstrated that trauma-hemorrhage also increases IL-1 and TNF-␣ release by Kupffer cells
but not by splenic and peritoneal macrophages. As a consequence of the initial injury, hepatic inflammation develops,
involving Kupffer cell activation, the release of cytokines and
chemokines, and, subsequently, neutrophil recruitment to the
liver. Hepatic dysfunction following ischemia-reperfusion is
also believed to be the result of subsequent activation of
Kupffer cells, which are the predominant source of oxygen free
radicals (23). Oxygen free radicals also stimulate cytokine
release and adhesion molecule expression following traumahemorrhage, which are events that also promote neutrophil
infiltration (25). The postulated mechanism of hepatic damage
is through the recruitment and activation of neutrophils and
oxidative injury, all leading to cell and tissue damage (35). Our
previous study (25) also demonstrated that the addition of a
free radical scavenger, 2-mercaptopropionyl glycine, to the
resuscitation fluid can decrease hepatocyte enzyme levels and
improve hepatic recovery. With regard to ICAM-1, however,
additional studies are needed to elucidate the ICAM-1-dependent events that lead to subsequent neutrophil infiltration following Fx-TH and whether such infiltration actually contributes to tissue injury.
The innate inflammatory response involved an exquisite
mechanism of autoregulation in the hepatocyte. The proinflammatory response is accompanied by a compensatory antiinflammatory reaction, as indicated by increased IL-10 production (19). IL-10 is an inhibitor of cytokine synthesis, and it
inhibits the production of proinflammatory cytokines such as
TNF-␣ and IL-6. The present results showed that Fx-TH not
only significantly increased proinflammatory cytokines
(TNF-␣ and IL-6) in hepatocyte extracts but also significantly
decreased levels of the anti-inflammatory cytokine IL-10 in
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Fig. 5. ICAM-1 protein levels in hepatocytes at 24 h after Fx-TH. ICAM-1
protein levels in hepatocytes were detected by Western blot analysis using
anti-ICAM-1 antibody. Blots (top) were analyzed densitometrically, and values are shown in the bar graph (bottom). *P ⬍ 0.05, Fx-TH vs. sham animals
(by ANOVA and Fisher’s post hoc test).
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Fig. 4. ICAM-1 mRNA expression in hepatocytes. ICAM-1 mRNA was
extracted from young and middle-aged mice at 24 h after the surgical
procedure, and RT-PCR was performed. ICAM-1 mRNA expression in middle-aged mice was markedly observed after Fx-TH. Blots (top) were analyzed
densitometrically, and values are shown in bar graph (bottom). *P ⬍ 0.05,
Fx-TH vs. sham animals (by ANOVA and Fisher’s post hoc test).
middle-aged mice. Decreased IL-10 levels in hepatocyte extracts of middle-aged mice were associated with increased
proinflammatory cytokines. It appears that the alteration in
these cytokines in hepatocyte extracts might be related to the
degree of hepatocellular inflammation following Fx-TH. Furthermore, these middle age-associated alterations contributed
to the increased hepatic inflammatory response following FxTH. Thus, it appears that aging changes the balance of the
cytokine network in the liver following Fx-TH.
The posthemorrhagic inflammatory response in the liver is
characterized by increased leukocyte-endothelium interactions,
as observed in an intravital microscopic study (32). Neutrophil
infiltration was previously implicated to play a contributory
role in the later stages of liver injury in models of hemorrhageresuscitation and hepatic ischemia-reperfusion (23). Transendothelial migration and the adherence of neutrophils to parenchymal cells requires the expression of adhesion molecules,
particularly ICAM-1. In models with severe sinusoidal endothelial cell damage, e.g., ischemia-reperfusion, blockade of
ICAM-1 was only moderately protective mainly because neutrophils have direct access to hepatocytes and neutrophilhepatocyte interactions are only partially dependent on
ICAM-1 (36). ICAM-1 is normally expressed at a low basal
level, but its expression can be enhanced by various inflammatory mediators such as TNF-␣ and IL-1␤ (44). Furthermore,
ICAM-1 can be induced by proinflammatory cytokines in
endothelial cells as well as hepatocytes (37).
A clinical study (44) has indicated the strong correlation
between circulating ICAM-1 levels and the development of
liver dysfunction during the course of sepsis, as characterized
by the highest serum bilirubin levels. In addition, the elevation
of ICAM-1 precedes the increase in bilirubin in septic patients.
However, the source of increased circulating ICAM-1 in producing liver damage remains unclear. Since circulating
ICAM-1 levels correlated with serum bilirubin levels and the
expression of ICAM-1 on hepatocytes correlates with the
EXPRESSION OF ICAM-1 IN THE LIVER FOLLOWING TRAUMA-HEMORRHAGE
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suggesting that ICAM-1 may be causally involved in the
development of liver dysfunction following trauma-hemorrhage. The results also indicate that there are divergent responses in mice of age groups that correspond to pediatric and
adult patient populations in humans. Our data thus support the
concept that the differences in gene and protein expression in
pediatric and adult patient populations may be responsible for
preventing or producing hepatocellular damage following
Fx-TH.
ACKNOWLEDGMENTS
We thank Shellie Hyde for the excellent technical assistance.
GRANTS
This work was supported by National Institute of General Medical Sciences
Grant R01-GM-37127.
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Surgical and traumatic injuries induce a systemic endocrinemetabolic response that includes the stimulation of the hypothalamic-pituitary-adrenal axis and sympathetic nervous system (3, 8). Our studies (3, 11, 47) on gender dimorphism have
indicated immunomodulatory effects of sex hormones as playing a critical role. It has been well documented that females
have a survival advantage over males and that testosterone
depletion/antagonism prevents immune suppression after
trauma and shock (3). Whether there are age-associated
changes in sex steroids that may contribute to the effects
observed in the present study was not investigated. Since aging
is associated with many endocrine alterations, it seems possible
that some of these changes will have an impact on cytokine
production and immune functions. Previous studies from our
laboratory (26, 27) have shown that high circulating levels of
estrogen in young females protect against the depression in
immune functions following trauma-hemorrhage and that the
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middle age-associated changes in hepatocyte extracts following trauma-hemorrhage are deleterious or not remains to be
examined in further studies. Nonetheless, the observed changes
in hepatocyte extracts have shown significant increases in
inflammatory mediators. These, together with an increase in
ICAM-1 in middle-aged mice following Fx-TH, may lead to
the severe inflammation in middle-aged male mice under those
conditions.
In conclusion, our present study demonstrates that middle
age influences liver cytokine production and ICAM-1 expression following Fx-TH. We found strong expression of ICAM-1
in hepatocytes of middle-aged mice during the study period,
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