Departments of 1 Surgery and 2 Pathology, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908
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ABSTRACT |
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A central role for nuclear
factor-B (NF-
B) in the induction of lung inflammatory injury is
emerging. We hypothesized that NF-
B is a critical early regulator of
the inflammatory response in lung ischemia-reperfusion injury, and
inhibition of NF-
B activation reduces this injury and improves
pulmonary graft function. With use of a porcine transplantation model,
left lungs were harvested and stored in cold Euro-Collins preservation
solution for 6 h before transplantation. Activation of NF-
B
occurred 30 min and 1 h after transplant and declined to near
baseline levels after 4 h. Pyrrolidine dithiocarbamate (PDTC), a
potent inhibitor of NF-
B, given to the lung graft during organ
preservation (40 mmol/l) effectively inhibited NF-
B activation and
significantly improved lung function. Compared with control lungs
4 h after transplant, PDTC-treated lungs displayed significantly
higher oxygenation, lower PCO2, reduced mean
pulmonary arterial pressure, and reduced edema and cellular
infiltration. These results demonstrate that NF-
B is rapidly
activated and is associated with poor pulmonary graft function in
transplant reperfusion injury, and targeting of NF-
B may be a
promising therapy to reduce this injury and improve lung function.
pyrrolidine dithiocarbamate; ischemia; organ preservation
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INTRODUCTION |
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RECENT ADVANCEMENTS IN LUNG transplantation have led to the increasing use of this life-saving operation for more patients with various end-stage pulmonary diseases. However, despite these improvements, difficulties with maintaining lung graft viability remain, and severe graft dysfunction occurs in 10-20% of lung transplant recipients (17). It has become increasingly evident that reperfusion after ischemia is responsible for the majority of tissue injury in lung transplantation. In addition, recent evidence confirms that lung ischemia-reperfusion injury is a result of the activation of inflammatory mediators in the early phase of reperfusion (7, 8, 18, 19).
The transcription factor nuclear factor-B (NF-
B) regulates the
expression of many genes in which early response products are critical
for the development of acute inflammation. NF-
B is composed of
various homo- or heterodimeric combinations of NF-
B/Rel proteins,
and different NF-
B dimers have varying transcriptional activation
properties. In unstimulated cells, NF-
B is retained in the cytoplasm
and bound to inhibitory proteins of the I
B family including
I
B-
. In response to inflammatory stimuli, including tumor
necrosis factor-
, interleukin-1
, and reactive oxygen species, I
B-
is phosphorylated by specific kinases and degraded by
proteasomes. Degradation of I
B-
releases NF-
B and allows
translocation to the nucleus, where NF-
B binds specific promoter
elements and induces gene transcription (2, 12). NF-
B
acts on target genes for proinflammatory cytokines, chemokines,
immunoreceptors, cell adhesion molecules, acute phase proteins, and
inducible nitric oxide synthase. The activation of NF-
B therefore
leads to a coordinated increase in the transcription of many genes in
which products mediate inflammatory responses. In addition, products of
the genes that are regulated by NF-
B often cause the activation of
NF-
B, which creates a positive feedback loop that may amplify and
perpetuate local inflammatory responses (2).
The inflammatory products of NF-B activation have been demonstrated
in several cellular models (22), and a critical role of
NF-
B in the inflammatory cascade in vivo is now emerging (3, 5, 13-15). In fact, several studies suggest that the
nuclear translocation of NF-
B is a prerequisite for the full
development of lung inflammatory injury (14).
Ischemia-reperfusion injury is also reported to be influenced by the
activation of NF-
B in the heart (5) and liver
(23). Reperfusion of ischemic tissues rapidly produces
reactive oxygen metabolites, which activate NF-
B and increase its
inflammatory products (21), all of which exacerbate tissue
reperfusion injury. Whereas these patterns of gene expression can be
correlated with tissue damage in models of endotoxin-induced lung
injury (3), immune complex-induced lung inflammation
(13), and warm ischemia-reperfusion injury (5,
23), it is unclear what influence NF-
B has on reperfusion
injury after ischemic lung storage in a clinical transplant
setting. In the current study, we investigated the importance of
NF-
B in a survival porcine model of left lung transplantation. We
hypothesized that NF-
B is a critical early regulator of the
inflammatory response in lung ischemia-reperfusion injury and that
inhibition of NF-
B activation reduces this injury and improves
pulmonary graft function.
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METHODS |
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Lung harvest.
Domestic swine of both sexes (25-30 kg) were randomly assigned to
two groups (n = 6 per group). Each animal was
anesthetized intramuscularly with xylazine (1 mg/kg), telazol (6 mg/kg), and atropine (0.01 mg/kg). After endotracheal intubation, the
pigs were ventilated with a volume-cycled ventilator at a tidal volume of 15 ml/kg, a respiratory rate of 15-20 breaths/min, and an
inspiratory oxygen concentration of 1.0. Anesthesia was maintained with
halothane, and each pig was anticoagulated with heparin sodium (400 U/kg). After a standard left thoracotomy, the pulmonary artery (PA) was dissected and the inferior pulmonary ligament was transected. After
pulmonary arterial injection of prostaglandin E1 (10 µg/kg), the main PA was clamped and cannulated. In our control group, 50 ml/kg of cold (4°C) Euro-Collins preservation solution (Fresenius) was infused into the PA from a height of 30 cm. Topical cooling was
achieved with a cold saline solution slush. The experimental group was
identical to the control group except that the Euro-Collins preservation solution was altered by the addition of pyrrolidine dithiocarbamate (PDTC; Sigma), an antioxidant and potent inhibitor of
NF-B activity, at 40 mmol/l. This dose was chosen based on studies
in rats where PDTC was injected at 150-200 mg/kg body wt (5,
15). The lung was then excised, prepared for implantation ex
vivo, immersed in cold Euro-Collins solution, and stored at 4°C for
6 h. Thus lungs in the experimental group were exposed to PDTC
during this 6-h storage period only. All animals received humane care
in compliance with the Guide for the Care and Use of Laboratory
Animals, published by the National Institutes of Health (NIH
publication no. 85-23, revised 1985).
Recipient preparation and transplantation procedure. In size-matched swine, anesthesia was maintained with isoflurane, and the animals were heparinized. Through a left thoracotomy, the left lung hilum was dissected; dissection around the recipient bronchus was minimized. The PA and bronchus were clamped, the pulmonary veins were ligated, and the native left lung was excised. Implantation commenced with the bronchial anastomosis followed by the pulmonary arterial and left atrial anastomoses. The vascular clamps were removed, a chest tube was inserted, and the chest was closed in layers.
Animals were allowed to survive for various time periods and were then euthanized by anesthetic overdose. Lung tissue from the control group was collected at 0 min, 30 min, 60 min, 4 h, and 20 h of reperfusion, and tissue from the experimental (PDTC) group was collected at 0 min, 30 min, 60 min, and 4 h of reperfusion (n = 6 per group per time period). In addition, normal nontransplanted pig lung tissue was sampled. Peripheral lung tissues were snap-frozen in liquid nitrogen and stored atLung physiological data collection.
For both control and PDTC groups (n = 6 for each), lung
graft function was analyzed at 4 h of reperfusion. At the time of study, a 16-gauge catheter was placed in the carotid artery for pressure measurements and blood gas analysis, a Cordis introducer was
placed in the internal jugular vein, and a Swan-Ganz catheter was
floated into the pulmonary artery. Carotid artery, central vein, and PA
pressures were recorded. The airflow and blood flow to the native right
lung were excluded to exclusively study the newly transplanted left
lung. A Fogarty balloon was placed into the right bronchus under
bronchoscopic vision, the right PA branches were ligated through the
reopened left thoracotomy, and the animal was allowed to stabilize for
30 min. Tidal volume, expiratory minute volume, and airway pressure
were displayed on the ventilator (Servo ventilator 900C,
Siemens-Elema), and dynamic compliance was then calculated. Mean PA
pressure (PAP), pulmonary capillary wedge pressure (PCWP), and cardiac
output (CO) were measured, and pulmonary vascular resistance (PVR) was
calculated using the equation PVR = [(PAP PCWP)/CO × 80]. In addition, blood gas measurements were obtained under
conditions of isolated transplant lung ventilation.
Nuclear protein extraction.
Nuclear extracts of whole lung tissues were prepared by the method of
Deryckere and Gannon (6). Briefly, frozen peripheral lung
tissue (0.5 g) was homogenized at 4°C with a Dounce tissue grinder in
solution A [0.6% Nonidet P-40, 150 mM NaCl, 10 mM HEPES, pH 7.4, 1 mM EDTA, 0.4 mM phenylmethylsulfonyl fluoride (PMSF), 4 µg/ml pepstatin A, 4 µg/ml leupeptin, and 4 µg/ml aprotinin]. The homogenate was centrifuged for 1 min at 1,500 rpm, and the remaining supernatant was collected and centrifuged for 5 min at 5,000 rpm. The pelleted nuclei were resuspended at 4°C in 300 µl of
solution B (25% glycerol, 20 mM HEPES, pH 7.4, 840 mM NaCl, 1.2 mM MgCl2, 0.2 mM EDTA, 0.5 mM dithiothreitol, 0.4 mM
PMSF, 4 µg/ml pepstatin A, 4 µg/ml leupeptin, and 4 µg/ml
aprotinin) and incubated on ice for 20 min. Samples were centrifuged,
and the supernatant (nuclear protein) was dialyzed at 4°C in
solution C (20 mM HEPES, pH 7.4, 100 mM NaCl, 0.2 mM EDTA,
and 25% glycerol) and stored at 80°C. Protein concentrations were
determined using Coomassie Plus Protein Assay Reagent (Pierce).
Electrophoretic mobility shift assay.
Double-stranded consensus oligonucleotide (5'-GTGAGGGGACTTTCCCAGGC-3',
Promega) was end labeled with [-32P]ATP (6,000 Ci/mmol; Amersham). Binding reactions containing equal amounts of
nuclear protein (5 µg) and 1.5 fmol of labeled oligonucleotide were
incubated for 30 min at room temperature in binding solution [10 mM
HEPES, pH 7.4, 50 mM KCl, 0.2 mM EDTA, 2.5 mM dithiothreitol, 150 µg/ml poly(dI-dC), and 0.05% Nonidet P-40]. Reaction products were
separated in a 6% nondenaturing polyacrylamide gel and analyzed by
autoradiography. Autoradiography and quantitation of autoradiographic
signals were performed using a PhosphorImager and ImageQuant software
(Molecular Dynamics). For supershift analysis, antibodies to NF-
B
p65 and p50 subunits were purchased from Santa Cruz Biotechnology.
Western blot analysis.
Frozen peripheral lung samples (0.5 g) were homogenized in 50 mM
Tris · HCl, pH 7.4, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM PMSF, 4 µg/ml pepstatin A, 4 µg/ml leupeptin, and 0.1% 2-mercaptoethanol and centrifuged at 4°C at 5,000 rpm. Protein concentrations were determined using Coomassie Plus Protein Assay Reagent (Pierce). Western
analysis of IB-
was performed using the PhosphoPlus I
B-
Antibody Kit (New England Biolabs). Briefly, samples (75 µg) were
separated in a denaturing 7.5% polyacrylamide gel and transferred to
nitrocellulose membrane. The membrane was incubated with primary
I
B-
antibody (1:1,000, rabbit polyclonal IgG) overnight at 4°C,
washed, and incubated with horseradish-conjugated secondary antibody
(1:2,000) for 1 h. Proteins were detected by incubating the
membrane with LumiGLO as instructed. Quantitation of autoradiographic signals was performed using the IS-1000 digital imaging system (Alpha Innotech).
Histological assessment. After harvest, representative blocks from the resected lungs were processed through Formalin fixation and routine paraffin embedding. Five-micrometer sections were stained with hematoxylin and eosin. Randomly coded sections from control vs. treated transplanted lungs as well as normal lungs directly harvested without any transplantation were submitted to a pulmonary pathologist for an assessment of "lung injury," if any, and if present a relative severity of injury. After a preliminary survey of the slides, lung injury was defined as the presence of increased interstitial cellularity within pulmonary parenchyma and/or extension of the cellular infiltrates into the alveolar spaces. The cells most commonly represented in these infiltrates were almost always segmented neutrophils with some additional macrophage infiltrates. Variable pulmonary edema in the form of intra-alveolar proteinaceous material was also assessed. These histological features were the basis for ranking the slides into three conceptual groups: normal, i.e., having no evidence of any increased cellular infiltrates or edema; mildly involved, i.e., having patchy or focal areas of abnormality involving less than 25% of the sampled histological area; and a third group that exhibited diffuse involvement (greater than 30% of the lung area involved), with denser interstitial infiltrates, more prominent intra-alveolar cellular infiltrates, and more prominent alveolar edema.
Wet-to-dry weights. For wet-to-dry weight ratios, parenchymal samples of whole lung tissue were blotted and weighed. After baking in a vacuum oven for 24 h at 50°C, tissues were then weighed to obtain dry weights. Tissues were again weighed another 24 h later to verify that complete dehydration occurred. Data were calculated as wet weight divided by dry weight and used as an indicator of pulmonary edema as illustrated by an increase in the wet-to-dry weight ratio.
Lung myeloperoxidase.
Myeloperoxidase (MPO) assay was performed on lung samples to quantify
neutrophil sequestration. Tissue (0.5 g) was placed in 5 ml of 0.5%
hexadecyltrimethylammonium bromide (HTAB) in 50 mM potassium phosphate
solution (pH 7.4) and disrupted by homogenizing at 4°C. The solution
was centrifuged at 15,000 g for 15 min at 4°C, and the
supernatant was discarded. The pellet was resuspended in 2 ml of 0.5%
HTAB in 50 mM potassium phosphate solution (pH 6.0) and homogenized.
Tissue was disrupted further by sonication and then underwent three
freeze-thaw cycles. The solution was then centrifuged at 15,000 g for 15 min at 4°C. Aliquots (0.1 ml) of supernatant were
added to 0.3 ml of assay buffer (0.1 mg/ml o-dianisidine,
0.7% H2O2, and 50 mM potassium phosphate, pH
6.0), and absorbance at 460 nm was measured over 2 min at room
temperature. MPO activity is expressed as change in absorbance per
milligram of wet lung weight per minute
(A460 · mg
1 · min
1).
Statistical analysis.
Statistical analysis was performed using analysis of variance on SPSS
software (SPSS, Chicago, IL), and Student's t-tests were
used to compare the two groups at various time points. Significant differences were determined using Tukey's honestly significant difference test. The data are expressed as the means ± SE. For quantitation of electrophoretic mobility shift assay (EMSA) gels, statistical analysis was performed on the raw data from the
PhosphorImager. Inasmuch as we present an increase of NF-B DNA
binding activity as a multiple increase, we do not include the
means ± SE of the raw data in RESULTS.
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RESULTS |
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Time course of NF-B DNA binding activity during lung reperfusion
after transplantation.
The time course of NF-
B activation (more precisely DNA binding
activity) during lung reperfusion was established by EMSA of nuclear
extracts from whole lung obtained at various time points after
initiation of reperfusion following transplantation. A low level of
NF-
B DNA binding activity was detected in the nuclei from normal
lungs that were not exposed to ischemia or reperfusion (Fig.
1A, lanes 1-2)
and this was not altered after cold ischemic storage alone (Fig.
1A, lanes 3-4). Nuclear activity of NF-
B increased dramatically with the onset of reperfusion, reaching maximal
levels by 30 min and maintaining high activation at 1 h (Fig.
1A, lanes 5-6 and 7-8,
respectively). NF-
B activity was eightfold higher after 30 min of
reperfusion and sixfold higher after 1 h compared with normal or
ischemic lung (P < 0.01 for both time points vs.
normal lungs). After 4 and 20 h of reperfusion, NF-
B DNA
binding activity approached baseline levels. Two representative samples
from each group for each time point are shown, and a small degree of
variability between animals was observed.
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Inhibition of NF-B nuclear activity by PDTC.
After demonstrating that in vivo reperfusion following lung
transplantation induces early NF-
B DNA binding activity, we next tested whether PDTC, an antioxidant and potent inhibitor of NF-
B activation (20), could suppress this activation.
Ischemia-reperfusion caused a significant increase in NF-
B DNA
binding activity at 30 and 60 min after the onset of reperfusion (Fig.
3, lanes 5-6 and
9-10, respectively). PDTC, which was administered to
the transplanted lung only during organ preservation (see
METHODS), dramatically inhibited NF-
B activation during
reperfusion (Fig. 3, lanes 7-8 and
11-12). In transplanted lungs that received
PDTC-enhanced Euro-Collins preservation solution, the nuclear
activation of NF-
B was reduced by 75% at 30 min and 68% at 60 min
of reperfusion compared with normal lungs. PDTC did not alter NF-
B
binding activity at other time points.
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Change in lung content of IB-
.
The protein level of I
B-
in lung tissue during reperfusion was
measured by Western analysis to determine whether the activation of
NF-
B was the result of the degradation of its inhibitory protein I
B-
. In whole lungs, I
B-
protein levels showed early
evidence of reduction during reperfusion after ischemic storage (Fig.
4). After 30 min of reperfusion (Fig. 4,
lanes 3-4), when NF-
B activation was maximal, the
level of I
B-
protein was reduced 70% from that of normal,
unoperated lungs (Fig. 4, lanes 1-2). The
inhibitory protein remained low throughout the 4-h reperfusion period.
Thus during the course of the lung inflammatory response, there was loss of I
B-
protein as assessed by Western analysis. Whereas the
addition of PDTC to the preservation solution inhibited the activation
of NF-
B (see Fig. 3), PDTC did not alter the protein expression of
I
B-
compared with control transplants (Fig. 4, lanes
9-12). I
B-
levels in the PDTC group were reduced at 30 min (Fig. 4, lanes 3-4) and 1 h (Fig.
4, lanes 5-6) of reperfusion by 72 and 78%,
respectively, from normal levels.
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Improvement in pulmonary graft function with PDTC.
We determined the influence of NF-B and the effects of
inhibition of its activity on lung graft function following
transplantation. Four hours after the initiation of reperfusion, we
isolated and studied the physiology of control transplant lungs and
transplant lungs that had received PDTC (Fig.
5). Transplanted lungs that were treated
with PDTC displayed a significant improvement in arterial oxygenation
and ventilation. The arterial PO2 of the PDTC
lungs was significantly higher than that of control lungs at 4 h
of reperfusion (334.7 ± 34.3 vs. 88.4 ± 7.9 mmHg,
P < 0.001; Fig. 5A), and the
PCO2 of PDTC lungs was significantly lower than that of control lungs (49.7 ± 10.4 vs. 96.9 ± 10.4 mmHg,
P = 0.005; Fig. 5B). At the conclusion of
the 4-h reperfusion period, PDTC significantly lowered the mean PAP to
where the mean PAP was 42.5 ± 4.7 mmHg in PDTC lungs and
55.2 ± 3.2 mmHg in control lungs (P = 0.02; Fig.
5C). In addition, the dynamic airway compliance of
the PDTC-treated lungs tended to be higher than that of control lungs (7.2 ± 0.8 vs. 5.6 ± 0.9 ml/cmH2O,
P = 0.10), although this was not found to be
significant (Fig. 5D).
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Histology. As described in METHODS, histological evaluation of lung injury was assessed in control transplant lungs after 4 h of reperfusion and compared with normal lungs and with transplanted lungs treated with PDTC during preservation. The pulmonary pathologist was informed only of the existence of three animal groups and had otherwise no knowledge of any of the other results reported herein. In a masked fashion, slides were then categorized into three groups, and the results are as follows: five of six lungs displaying the most diffuse involvement of abnormality and infiltration were correctly identified as control transplant lungs, four of six lungs displaying mildly involved abnormality were correctly identified as PDTC-treated transplanted lungs, and five of six lungs displaying no evidence of abnormality were correctly identified as normal lungs. One PDTC-treated lung was misidentified as normal, one PDTC-treated lung was misidentified as control transplant, one normal lung was misidentified as PDTC treated, and one control transplant was misidentified as PDTC treated. Critically, no normal lungs were misidentified as control transplant and vice versa.
Representative slides from each group are shown in Fig. 6. Histopathological specimens of the control transplant lungs after 4 h of reperfusion showed clear evidence of diffuse abnormality, with dense interstitial infiltrates and edema formation (Fig. 6, B and E). In the transplanted lungs that were treated with PDTC during preservation, the abnormality and cellular infiltration was only mild (Fig. 6, C and F) and closely resembled that of normal lung tissue (Fig. 6, A and D).
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Pulmonary edema.
As an indicator of pulmonary edema, wet-to-dry weight ratios were
calculated. As shown in Fig. 7,
wet-to-dry weight ratios were significantly increased in control
transplant lungs after 4 h of reperfusion compared with those in
pretransplanted normal lungs (P < 0.01). These
wet-to-dry weight ratios were significantly reduced in transplanted
lungs that were treated with PDTC during preservation
(P < 0.01, Fig. 7).
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Lung MPO activity.
We utilized the MPO biochemical assay to measure tissue
neutrophil infiltration over the course of in vivo reperfusion and its
correlation with NF-B activation. Tissue MPO activity in control
transplant lungs was significantly elevated after 30 min of reperfusion
compared with that in normal lung tissue and remained significantly
elevated throughout the 4 h of reperfusion (10.8 ± 1.8
A460 · mg
1 · min
1
after 30 min, P = 0.006, and 15.5 ± 1.4
A460 · mg
1 · min
1 after
4 h, P < 0.001 vs. 4.3 ± 1.5
A460 · mg
1 · min
1 for
normal lung). However, the administration of PDTC to the transplanted
lungs did not alter the tissue MPO activities compared with those in
the control lungs (i.e., the PDTC-treated lungs displayed a similar
elevation in MPO activity as did the control, untreated lungs). The MPO
activity in the PDTC-treated group was 13.2 ± 2.3
A460 · mg
1 · min
1 after
30 min (P = 0.46 vs. control lungs at 30 min), and
14.3 ± 2.2
A460 · mg
1 · min
1 after
4 h of reperfusion (P = 0.65 vs. control lungs at
4 h).
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DISCUSSION |
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During the past few years, many of the cellular and molecular
events mediating lung ischemia-reperfusion injury have been clarified
(17, 18). The inflammatory cascade has emerged as a
critical mechanism of lung reperfusion injury, but current therapeutic interventions that target this pathway have yet to demonstrate clear
clinical benefit. This is likely due to the fact that these therapies
mainly focus on inhibition of specific distal components of the
inflammatory response; however, inflammation is initiated and amplified
by many redundant and overlapping molecular pathways. Recently, NF-B
has emerged as a rapid response transcription factor that plays a
central role in the induction of lung inflammatory injury (2,
12). NF-
B is an amplifying and perpetuating mechanism that
can exaggerate the disease-specific inflammatory process through the
coordinated activation of many inflammatory genes. The role of NF-
B
in the transcriptional activation of key inflammatory genes has been
studied in numerous cell lines, and recent studies have demonstrated
the in vivo regulatory role of NF-
B in models of endotoxin-induced
lung injury (3), immune complex-induced lung
inflammation (13), systemic inflammation (11,
15), and warm ischemia-reperfusion injury (5).
In the present study, we have demonstrated for the first time that
NF-B is activated in vivo in a clinical lung transplant setting of
cold ischemia and reperfusion. After 6 h of ischemic organ storage
without reperfusion, the level of NF-
B nuclear activation is not
increased from that of normal lung tissue (see Fig. 1A).
Shortly after initiation of reperfusion, NF-
B activity increases
dramatically and peaks in our model at 30 min and maintains high
activity at 1 h of reperfusion. This pattern clearly indicates that the activation of NF-
B depends entirely on events occurring during the early phases of reperfusion, a finding also suggested in
models of warm ischemia-reperfusion (5, 23). Because whole tissues were used in our study, we cannot speculate at this time what
cell type or types are involved in the upregulation of NF-
B. It has
been shown that many cell types are capable of upregulating NF-
B,
and further studies will be needed to address this question. Reperfusion of ischemic tissue has been shown to be associated with a
rapid burst of reactive oxygen metabolites. These free radicals have
been shown previously to activate NF-
B in a number of experimental
systems (21) and likely contribute to the early rise in
NF-
B activity seen in our lung transplant model. We observed the
preferential increase in the fastest migrating NF-
B complex during
reperfusion and have shown by supershift analysis that this complex is
composed of p65 NF-
B protein, most likely in a homodimeric form.
Homodimers of p65 protein comprise a specific subset of NF-
B
complexes and have been shown to be a transcriptionally active complex
for several inflammatory genes such as intercellular adhesion
molecule-1, interleukin-8, and tissue factor (1). Of the other two (slower migrating) NF-
B complexes observed in pig
lung by EMSA (Fig. 1), the slowest migrating complex also appeared to
be supershifted by the p65 antibody (Fig. 2). Thus this complex may
also be composed of NF-
B p65 protein but in a different
configuration than the smallest migrating complex. The intermediate
migrating complex did not supershift with antibody to either p50 or p65
and thus may be composed of a different NF-
B protein such as p52
(NF-
B2), Rel B, or C-Rel. Alternatively, it is possible
that the p50 antibody that we used may not be immunoreactive to pig antigen.
The increase in NF-B activity is associated with poor graft function
in our study of lung transplants. After the rise in NF-
B in the
control transplant lungs, these lung grafts exhibit poor oxygenation,
poor ventilation (PCO2), elevated mean PAP, and
reduced airway compliance. On histopathological specimens, the lungs
demonstrate alveolar and interstitial edema. In addition, a dramatic
increase in tissue neutrophil infiltration is evident in histological
specimens. Several studies have described the upregulation of adhesion
molecule expression (1) and cytokine-induced neutrophil
chemoattractant mRNA expression (3) associated with NF-
B activation, all of which may contribute to the neutrophilic inflammation associated with NF-
B.
To further clarify the connection between NF-B and in vivo lung
reperfusion injury, we treated the lung graft with PDTC, a potent
inhibitor of NF-
B activation, during organ preservation. A number of
studies have demonstrated the inhibition of NF-
B with PDTC in vitro
(16, 20), and recent reports have justified its use in
vivo (10, 11, 15). However, treatment of a donor organ
with PDTC at the time of harvest and ischemic preservation before
transplantation have yet to be described. We have shown that this
strategy inhibited the activation of NF-
B after lung transplant and
reperfusion (see Fig. 3). At the peak of NF-
B activity 30 min after
reperfusion, PDTC reduced this level by 75%. The inhibition of NF-
B
nuclear activity improved the physiology of the transplanted lung, with
the PDTC-treated lungs demonstrating significantly higher levels of
PO2, lower PCO2, and
decreased mean PAP, whereas there was a trend toward improved airway
compliance. In addition, histological analysis of the PDTC-treated
lungs closely resembled that of normal lungs, whereas the nontreated
lungs displayed high levels of cellular infiltration and edema. Our
results indicate that NF-
B is critically important in lung
reperfusion injury after transplantation and is an important regulator
of neutrophilic infiltration.
The dithiocarbamates, such as PDTC, represent a class of antioxidants
reported to be potent inhibitors of NF-B and are capable of
inhibiting the inflammatory process associated with the activation of
NF-
B. The most effective inhibitor of NF-
B appears to be the
pyrrolidine derivative of dithiocarbamate (PDTC) as a result of its
ability to traverse the cell membrane and its prolonged stability in
solution at physiological pH (24). PDTC, as well as other
antioxidants, may inhibit NF-
B by suppressing the production of
intracellular reactive oxygen species. Another chemical property of
PDTC is its chelating activity for heavy metals, which is the reason
that the diethyl derivative is used for the treatment of heavy metal
poisoning. Because nonthiol metal chelators such as o-phenanthroline and desferrioxamine can also inhibit
NF-
B activation, it is possible that the inhibitory effect of PDTC
relies on both of its properties (20). Thus PDTC may have
other cellular effects as yet undefined, but it is well established
that inhibition of NF-
B is a major mechanism of the
anti-inflammatory actions of PDTC.
We were unable to correlate MPO activity with the decreased neutrophil infiltration (inflammation) observed histologically. Histological evaluation by a pulmonary pathologist clearly indicated significant interstitial cellularity within pulmonary parenchyma and/or extension of the cellular infiltrates into the alveolar spaces of transplanted lungs. The cells most commonly represented in these infiltrates were almost always segmented neutrophils, with some additional macrophage infiltrates. These infiltrates were greatly reduced in histological specimens from PDTC-treated transplanted lungs, but the MPO activities were not reduced. The MPO assay gives no information about the location or extent of neutrophil sequestration, and it is possible that neutrophils may accumulate in nonalveolar regions of the lung such as the airways. A more plausible explanation is that our MPO activity is based on wet weight of the lung samples (see METHODS), and we have shown that significant edema occurs in the control lungs (Fig. 7), which would result in blunted MPO activities in these samples. In addition, the MPO assay can be greatly affected by the presence of whole blood (hemoglobin) in tissues, and the lungs were not perfused with saline to wash out blood remaining in the pulmonary circulation. We do not question the histological evaluation of neutrophil infiltration in our samples, but the lack of correlation with MPO activity indicates that a more rigorous application of MPO studies is required for accurate quantitation of neutrophil infiltration by MPO activity.
The activation of NF-B involves removal of the inhibitory subunit
I
B-
from a latent cytoplasmic complex. Various stimuli first
phosphorylate and then degrade I
B-
, allowing free NF-
B to
translocate to the nucleus where it binds to specific promoter sequences and initiates inflammatory gene transcription
(9). Several I
B proteins have been identified, but the
degradation of I
B-
has shown the highest correlation with the
activation of NF-
B (25). Our findings in a lung
transplant model support a role for I
B-
as an inhibitor of
NF-
B because activation of NF-
B during lung reperfusion injury
was accompanied by the reduction of I
B-
protein soon after the
initiation of reperfusion. Interestingly, the inhibition of NF-
B by
PDTC does not appear to be regulated by I
B-
. In our study, PDTC
inhibited NF-
B activation but not by stabilizing I
B-
. Whereas
some studies have suggested that the mechanism of PDTC is through the
blockade of phosphorylation of I
B-
(25), our in vivo
findings indicate that the inhibition of NF-
B by PDTC does not occur
through the preservation of I
B-
. Instead, the mechanisms for the
effects of PDTC on NF-
B may involve inhibition of binding of the
transcription factor to DNA, as demonstrated by others (4,
16), rather than an effect on the activation process.
There is now abundant evidence showing that lung reperfusion injury
after transplantation is largely due to the activation and upregulation
of mediators of the inflammatory cascade, and the central role that
NF-B plays in the induction of lung inflammatory injury is now
emerging. We have shown for the first time that NF-
B is rapidly
activated and is associated with poor pulmonary graft function in
transplant reperfusion injury. PDTC given to the lung graft at the time
of organ preservation effectively inhibited the activation of NF-
B
and significantly improved lung function. One important question
remaining is whether PDTC treatment and subsequent NF-
B inactivation
have any long-term effect on organ survival and ultimate function.
Although this question cannot be addressed by these studies, it is
reasonable to speculate that there are positive long-term effects of
NF-
B inhibition on graft function. It has become increasingly
evident that reperfusion after ischemia is responsible for the majority
of tissue injury in lung transplantation, and those lungs that might
otherwise undergo severe graft dysfunction following transplant might
be prevented from doing so by intervention with NF-
B inhibitors such
as PDTC. Whereas the mechanisms of action of PDTC continue to be
investigated and new inhibitors of NF-
B continue to be developed, we
believe that a therapeutic strategy directed at the inhibition of
NF-
B activation within the transplanted lung may prove effective in
reducing lung ischemia-reperfusion injury.
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ACKNOWLEDGEMENTS |
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We thank Anthony J. Herring for technical assistance.
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FOOTNOTES |
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This work was supported by National Heart, Lung, and Blood Institute Grant R01-HL-56093 and National Research Service Award Fellowship F32-HL-09820-01A1. The research also was supported by National Institute of Child Health and Human Development Grant HD-28934, through cooperative agreement U54 as part of the Specialized Cooperative Centers Program in Reproduction Research.
Address for reprint requests and other correspondence: V. E. Laubach, Dept. of Surgery, PO Box 801359, Univ. of Virginia Health Sciences Center, Charlottesville, VA 22908 (E-mail: vel8n{at}virginia.edu).
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.
Received 30 September 1999; accepted in final form 15 May 2000.
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