Department of Surgery, University of Louisville School of Medicine, Louisville, Kentucky 40292
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ABSTRACT |
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Hepatic
ischemia and reperfusion cause local and remote organ injury.
This injury culminates from an integrated cascade of proinflammatory
cytokines, chemokines, and adhesion molecules, many of which are
regulated by the transcription factor nuclear factor-B (NF-
B).
The anti-inflammatory cytokine interleukin-10 (IL-10) has been shown to
have inhibitory effects on NF-
B. The objective of the current study
was to determine whether IL-10 could suppress pulmonary NF-
B
activation and ensuing lung injury induced by hepatic
ischemia-reperfusion. C57BL/6 mice underwent partial hepatic
ischemia with or without intravenous administration of IL-10.
Hepatic ischemia-reperfusion resulted in pulmonary NF-
B activation, increased mRNA expression of tumor necrosis factor-
(TNF-
), and macrophage inflammatory protein-2 (MIP-2), as well as
increased pulmonary neutrophil accumulation and lung edema. Administration of IL-10 suppressed lung NF-
B activation, reduced TNF-
and MIP-2 mRNA expression, and decreased pulmonary neutrophil recruitment and lung injury. The data suggest that IL-10 protects against hepatic ischemia and reperfusion-induced lung injury by inhibiting lung NF-
B activation and the resulting pulmonary
production of proinflammatory mediators.
inflammation; tumor necrosis factor-; neutrophils; mice; nuclear
factor-
B
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INTRODUCTION |
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HEPATIC ISCHEMIA-REPERFUSION injury is a complication
of liver resectional surgery, liver transplantation, and hemorrhagic shock with fluid resuscitation and may lead to local and remote organ
damage (17). There is a preponderance of literature suggesting that the
pathogenesis of the resulting tissue injury in remote organs, such as
the lung, is related to the hepatic generation of the proinflammatory
cytokine tumor necrosis factor- (TNF-
). After hepatic
ischemia and reperfusion, TNF-
initiates a mediator cascade
in the lung, including upregulation of the C-X-C chemokines, epithelial
neutrophil-activating protein ENA-78, macrophage inflammatory protein-2
(MIP-2), and KC, as well as increased pulmonary vascular expression of
intercellular adhesion molecule-1 (ICAM-1) (3-5, 26).
The coordinated effects of C-X-C chemokines and adhesion molecules
result in pulmonary neutrophil accumulation and ensuing lung injury.
The gene expression of these proinflammatory mediators, including
TNF-
, C-X-C chemokines, and ICAM-1, is controlled at least in part
by the transcription factor nuclear factor-
B (NF-
B) (2, 7, 24,
25). Although activation of NF-
B occurs in liver during
ischemia-reperfusion injury (28), it is unknown whether NF-
B
is activated in remote organs or whether remote organ NF-
B
activation is important for the development of tissue injury.
Recent reports have demonstrated that the anti-inflammatory cytokine
interleukin-10 (IL-10) has hepatoprotective effects during liver
transplantation (27) and experimental liver injury induced by
galactosamine and lipopolysaccharide (18, 23). IL-10 has also been
shown to suppress experimental lung inflammatory injury induced by
intrapulmonary deposition of IgG immune complexes through inhibitory
effects on NF-B activation (15). In the current studies, we sought
to determine whether hepatic ischemia and reperfusion caused
NF-
B activation in the lung. In addition, we evaluated the effects
of IL-10 on the development of lung inflammatory injury induced by
hepatic ischemia and reperfusion.
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MATERIALS AND METHODS |
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Hepatic ischemia and reperfusion injury model. Male C57BL/6 mice (Charles River Laboratories, Wilmington, MA) weighing 22-28 g were used in all experiments. This project was approved by the University of Louisville Animal Care and Use Committee and was in compliance with the guidelines of the National Institutes of Health. The model of partial hepatic ischemia and reperfusion employed was prepared as described previously (16). Briefly, mice were anesthetized with pentobarbital sodium (60 mg/kg ip). Mice received either sterile saline or recombinant murine IL-10 (1 µg; R&D Systems, Minneapolis, MN) via the lateral tail vein before induction of ischemia. A midline laparotomy was performed, and an atraumatic clip was used to interrupt the arterial and the portal venous blood supply to the cephalad lobes of the liver. After 90 min of partial hepatic ischemia, mice again received either sterile saline or IL-10 (1 µg) via the lateral tail vein and the clip was removed, initiating hepatic reperfusion. Sham control mice underwent the same protocol but without vascular occlusion. Mice were killed after the indicated periods of reperfusion, and lung tissues and blood samples were taken for analysis.
Myeloperoxidase assay. Lung myeloperoxidase (MPO) content was assessed by methods described previously (26). Briefly, lung tissue (50 mg) was homogenized in 2 ml of homogenization buffer (3.4 mM KH2HPO4 and 16 mM Na2HPO4, pH 7.4). After centrifugation for 20 min at 10,000 g, 10 volumes of resuspension buffer (43.2 mM KH2HPO4, 6.5 mM Na2HPO4, 10 mM EDTA, and 0.5% hexadecyltrimethylammonium, pH 6.0) were added to the pellet and the samples were sonicated for 10 s. After heating for 2 h at 60°C, the supernatant was reacted with 3,3',5,5'-tetramethylbenzidine (Sigma Chemical, St. Louis, MO) and read at 655 nm.
Lung edema. The extent of lung edema was measured by tissue wet-to-dry weight ratios. After dissection, lung samples were weighed and then placed in a drying oven at 55°C until a constant weight was obtained. In this determination, lung edema is represented by an increase in the wet-to-dry weight ratio.
Reverse transcription-polymerase chain reaction.
Fresh lung samples were immediately frozen when the mice were killed.
The extraction of total RNA was performed using a commercially available kit (Qiagen, Valencia, CA). A 1-µg aliquot of total lung
RNA was reverse transcribed to cDNA using the Geneamp RNA polymerase
chain reaction (PCR) protocol (Perkin-Elmer, Norwalk, CT) with random
hexamers to prime the reverse transcription (RT) with an excess of
deoxyribonucleotides. The cDNA products were amplified with 2.5 units
of AmpliTaq DNA polymerase, 3 mM
Mg2+, and 0.5 mM primer. After 2 min of initial melting at 95°C, the mixture was amplified for a
total of 30 cycles with a three-step cycle process that began with
melting at 95°C for 60 s, annealing at 59°C for 90 s, followed
by extension at 72°C for 10 s. The final cycle was followed by 12 min of soaking at 72°C. Ten microliters of each RT-PCR were
electrophoresed in a 3.5% agarose (GIBCO) gel and stained with
ethidium bromide. RT-PCR amplification of a housekeeping gene
(-actin) was performed to verify equal loading of RNA and
cDNA in the RT-PCRs. Products of RT-PCRs were photographed.
NF-B activation.
Nuclear extracts of lung tissue were prepared by the method of
Deryckere and Gannon (10). Protein concentrations were determined by
bicinchoninic acid assay with trichloroacetic acid precipitation using
BSA as a reference standard (Pierce, Rockford, IL). Double-stranded NF-
B consensus oligonucleotide
(5'-AGTGAGGGGACTTTCCCAGGC-3'; Promega, Madison, WI) was end
labeled with
[
-32P]ATP (3,000 Ci/mmol at 10 mCi/ml; Amersham, Arlington Heights, IL). Binding
reactions containing equal amounts of protein (20 µg) and 35 fmol
(~50,000 counts/min, Cherenkov counting) of oligonucleotide were
performed for 30 min in binding buffer [4% glycerol, 1 mM MgCl2, 0.5 mM EDTA, pH 8.0, 0.5 mM
dithiothreitol, 50 mM NaCl, 10 mM Tris, pH 7.6, and 50 µg/ml
poly(dI-dC); Pharmacia, Piscataway, NJ]. Reaction volumes were
held constant to 15 µl. Reaction products were separated in a 4%
polyacrylamide gel and analyzed by autoradiography.
Statistical analysis. All data are expressed as means ± SE. Data were analyzed with a one-way analysis of variance, and individual group means were then compared with a Student-Newman-Keuls test. Differences were considered significant when P < 0.05. For calculations of percent change, negative control values were subtracted from positive control and treatment group values.
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RESULTS |
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Activation of NF-B in lung during hepatic
ischemia-reperfusion injury.
The time course of NF-
B activation in the lung during hepatic
ischemia-reperfusion injury was determined by electrophoretic mobility shift assays of lung nuclear extracts (Fig.
1A).
Autoradiograms were digitized and relative band intensity was
determined using image-analysis software (Fig.
1B). In lungs from sham-operated controls, there was little NF-
B in nuclear extracts. Unexpectedly, nuclear translocation (activation) of NF-
B was observed after 90 min
of hepatic ischemia but before reperfusion (0-h reperfusion). Lung NF-
B activation increased modestly over the 4-h time course studied, with maximal activation occurring 1 and 4 h after hepatic reperfusion.
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Effects of IL-10 on lung NF-B activation and mRNA
expression of TNF-
and MIP-2.
Because lung NF-
B activation induced by hepatic ischemia and
reperfusion was maximally activated within 1-4 h of hepatic reperfusion, we assessed the effects of exogenously administered IL-10
on lung NF-
B activation after 1 h of reperfusion (Fig. 2). As in previous experiments, lung
NF-
B activation was increased by hepatic
ischemia-reperfusion compared with sham controls. In mice
receiving intravenous administration of IL-10, lung NF-
B activation
was almost completely inhibited.
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Effects of IL-10 on lung neutrophil recruitment and injury.
We have previously shown in this model that pulmonary neutrophil
accumulation and lung injury correlate with lung mRNA and serum levels
of MIP-2 and TNF- (26). The recruitment of neutrophils to lung and
the development of lung injury occur after 3-4 h of hepatic
reperfusion (26). Therefore, we assessed whether the reduction in
TNF-
and MIP-2 mRNA expression by IL-10 was associated with
decreased recruitment of neutrophils and lung injury after 4 h of
hepatic reperfusion. Pulmonary neutrophil recruitment was determined by
lung MPO content. Hepatic ischemia and 4 h of reperfusion caused significant increases in lung MPO content compared with the sham
controls (Fig. 4). Intravenous
administration of IL-10 reduced lung MPO content by 61%
(P = 0.016). Lung edema, as measured by lung wet-to-dry weight ratio, was used as an index of lung injury.
Hepatic ischemia and 4 h of reperfusion induced significant lung edema (Fig. 5). Administration of
IL-10 suppressed increases in lung wet-to-dry weigh ratio by 72%
(P = 0.004).
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DISCUSSION |
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Hepatic injury induced by ischemia and reperfusion is
associated with two distinct phases. During the initial phase,
activated Kupffer cells cause liver injury by releasing reactive oxygen species (12). After this initial oxidant-induced injury, Kupffer cells
and hepatocytes produce proinflammatory cytokines, including TNF-,
which initiates a mediator cascade leading to hepatic neutrophil recruitment (6). The ensuing liver injury is mediated by oxidants and
proteases released by sequestered neutrophils (11). It has been shown
that activation of NF-
B occurs during the initial phase of hepatic
injury (28). It is thought that NF-
B activation may be responsible
at least in part for the increased hepatic production of
proinflammatory cytokines during this phase. Remote organ injury caused
by hepatic ischemia and reperfusion, such as that induced in
the lung, is thought to be a result of liver-derived TNF-
. In fact,
blockade of TNF-
using antibody neutralization greatly reduced
hepatic ischemia and reperfusion-induced lung inflammatory
injury in rats (6).
Unexpected were the findings that NF-B was activated in the lung
during the period of hepatic ischemia. The precise mechanism of
this effect is unknown, but serum TNF-
is not increased at this time
(Yoshidome and Lentsch, unpublished data). Our observations of
increased lung NF-
B activation during hepatic ischemia are consistent with those of a recent report demonstrating increased lung
production of C-X-C chemokines during hindlimb ischemia before reperfusion (1). In those studies, it was noted that there was systemic
activation of complement during the ischemic period. Although
complement activation products have been shown to augment pulmonary
production of TNF-
and C-X-C chemokines (9, 20), these effects are
not mediated by activation of NF-
B (9, 14). Alternatively, mediators
could be released by the nonischemic liver in response to increased
blood flow to that portion of the organ. However, there is no evidence
of tissue injury in nonischemic lobes (16).
On the basis of our findings, increased NF-B activation in lung
after hepatic ischemia-reperfusion appears to lead to pulmonary expression of TNF-
and MIP-2. Activation of NF-
B in alveolar macrophages has been shown to be required for the intrapulmonary production of TNF-
and MIP-2, as well as development of lung injury
induced by IgG immune complexes (13). IL-10 has been shown to suppress
NF-
B in alveolar macrophages by preventing degradation of the
NF-
B inhibitory protein I
B-
(15). In the present studies,
intravenous administration of IL-10 suppressed lung NF-
B activation
induced by hepatic ischemia and reperfusion. These effects were
associated with reduced lung expression of TNF-
and MIP-2 mRNAs,
both of which are required for full development of lung injury after
hepatic ischemia and reperfusion (3, 26). Reductions in TNF-
and MIP-2 corresponded to decreased lung neutrophil accumulation and
reduced lung edema. Additionally, IL-10 may reduce neutrophil
recruitment into the lung through effects on the pulmonary endothelium.
IL-10 has been shown to prevent upregulation of lung vascular ICAM-1
during lung inflammation (19). Furthermore, recent studies showed that
blockade of TNF-
decreases pulmonary vascular expression of the
adhesion molecule ICAM-1, resulting in reduced lung recruitment of
neutrophils (4). Like TNF-
and MIP-2, ICAM-1 is regulated at the
transcriptional level by NF-
B (7). Although not investigated in the
current study, it is likely that IL-10 reduces pulmonary vascular
expression of ICAM-1 either directly through inhibition of
NF-
B in endothelial cells or indirectly by suppression of
TNF-
production as a contributing mechanism for reduction of lung
neutrophil accumulation.
IL-10 has been shown to be beneficial in the setting of organ
transplantation, including liver (8, 27) and heart (21, 22); in both
cases, treatment with IL-10 increased allograft survival. The current
studies demonstrate that IL-10 may also be protective in surgical or
trauma-related organ injuries occurring secondary to hepatic
ischemia-reperfusion. IL-10 reduced lung injury induced by
hepatic ischemia and reperfusion. IL-10 decreased the extent of
lung neutrophil recruitment in association with reductions in lung
expression of TNF- and MIP-2 mRNAs. These effects of IL-10 were
accompanied by almost complete inhibition of NF-
B activation in the
lung. Thus the current studies suggest that the use of agents such as
IL-10 to target upstream components of the inflammatory response, such
as NF-
B, may have therapeutic applications for organ injury caused
by hepatic surgery, transplantation or hemorrhagic shock.
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ACKNOWLEDGEMENTS |
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This work was supported in part by grants from the Alliant Community Trust Fund and the Jewish Hospital Foundation.
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FOOTNOTES |
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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" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: A. B. Lentsch, Dept. of Surgery, Univ. of Louisville School of Medicine, J. G. Brown Cancer Center, Rm. 426, 529 South Jackson St., Louisville, KY 40292 (E-mail: ablent01{at}ulkyvm.louisville.edu).
Received 12 February 1999; accepted in final form 9 June 1999.
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