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B Kinase Expression in Airway Epithelium Generates Neutrophilic Lung Inflammation1
* Department of Medicine, Division of Allergy, Pulmonary and Critical Care Medicine, Departments of
Cell and Developmental Biology,
Biomedical Engineering, and
Cancer Biology, Vanderbilt University School of Medicine, Nashville, TN 37232; and
¶ Department of Veterans Affairs Medical Center, Nashville, TN 37212
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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B activation is sufficient to generate
lung inflammation in vivo, we selectively expressed a constitutively
active form of IB kinase 1 (cIKK1) or IB kinase 2 (cIKK2) in
airway epithelium. After intratracheal administration of adenoviral
vectors expressing cIKK1 or cIKK2 to transgenic reporter mice that
express Photinus luciferase under the control of an
NF-B-dependent promoter, we detected significantly increased
luciferase activity over time (up to 96 h). Compared with control
mice treated with adenoviral vectors expressing 
-galactosidase, lung
bioluminescence and tissue luciferase activity were increased in
NF-B reporter mice treated with adenovirus (Ad)-cIKK1 or
Ad-cIKK2. NF-B activation in lungs of Ad-cIKK1- and Ad-cIKK2-treated
mice was confirmed by immunoblots for RelA and EMSA from lung nuclear
protein extracts. Mice treated with Ad-cIKK1 or Ad-cIKK2 showed
induction of mRNA expression of several chemokines and cytokines in
lung tissue. In lung lavage fluid, mice treated with Ad-cIKK1 or
Ad-cIKK2 showed elevated concentrations of NF-B-dependent chemokines
macrophage-inflammatory protein 2 and KC and increased numbers
of neutrophils. Coadministration of adenoviral vectors expressing a
transdominant inhibitor of NF-B with Ad-cIKK1 or Ad-cIKK2 resulted
in abrogated NF-B activation and other parameters of lung
inflammation, demonstrating that the observed inflammatory effects of
Ad-cIKK1 and Ad-cIKK2 were dependent on NF-B activation by these
kinases. These data show that selective expression of IB kinases in
airway epithelium results in NF-B activation, inflammatory mediator
production, and neutrophilic lung inflammation. Therapies targeted to
NF-B in lung epithelium may be beneficial in treating inflammatory
lung diseases. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B, along with AP-1, STAT, and C/EBP family
transcription regulatory factors, coordinates the activation of many
genes involved in host defense and inflammatory responses in the lungs.
NF-B activation is involved in the pathogenesis of a variety of
inflammatory lung diseases including asthma, cystic fibrosis,
pneumonia, and the acute respiratory distress syndrome, and may have a
role in idiopathic pulmonary fibrosis and environmental lung diseases
(1, 2). Although activation of NF-B is necessary for
maximal transcription of many adhesion molecules, enzymes, cytokines,
and chemokines important in the initiation of inflammation, the
independent effects of selective activation of this transcription
factor complex in the lungs are undefined.
Five members of the mammalian NF-B/Rel family have been identified.
These include NF-B1 (p50/p105), NF-B2 (p52/p100), RelA (p65),
RelB, and cRel. These proteins form homodimers or heterodimers and
share the conserved Rel homology domain, which is involved in
dimerization and DNA binding to specific cognate sequences. The
preponderant form of NF-B in most cells consists of a heterodimer
containing p50 and RelA (p65), which contains a transactivation domain
(1, 3). In quiescent cells, NF-B complexes are
sequestered in the cytoplasm by inhibitory proteins (IB-
,
IB-, IB-
) (4). Stimulation of the NF-B
pathway is mediated by diverse signal transduction cascades, resulting
in phosphorylation of IBs. Phosphorylated IB- (or IB-)
is ubiquitinated and degraded by the 26S proteasome, allowing nuclear
translocation of the NF-B complexes (5, 6).
The specific protein kinases responsible for IB phosphorylation have
been identified in a high molecular mass complex called the signalsome.
This protein complex contains two kinases (IB kinase
(IKK)31 and IKK2) that
have the ability to phosphorylate IB and structural/regulatory
subunits, including NF-B essential modifier (or IKK
), Cdc37, and
Hsp90 (7, 8, 9, 10, 11, 12, 13, 14). IKK1 and IKK2 have a similar primary
structure and form homo- or heterodimers. IKK2 appears to be the
principal kinase involved in IB phosphorylation resulting from cell
activation by the proinflammatory cytokines (TNF- and IL-1) and
bacterial LPS (15, 16, 17). The function of IKK1 in
generation of inflammation is less well defined; however, dimerization
and phosphorylation of specific serine residues of IKK2 by IKK1 are
required for kinase activity of the complex (18, 19). IKK1
appears to have an important role in development and may have
additional functions independent of NF-B (20).
In the present study, we hypothesized that expression of constitutively
active forms of IKK1 or IKK2 in airway epithelium would be sufficient
to induce an inflammatory response in the lungs. We and others have
shown that airway epithelial cells can be stimulated to activate
NF-B and produce cytokines and chemokines that are important for
directing innate immune responses in the lungs (21, 22, 23).
Mediator production by airway epithelial cells is thought to contribute
to a variety of lung inflammatory states.
Using direct intratracheal (IT) delivery of replication-deficient
adenoviral vectors that express constitutively active IKK1 (cIKK1),
IKK2 (cIKK2), or control vectors, we demonstrate that expression of
cIKK1 or cIKK2, but not -galactosidase (-gal), in lung epithelium
causes sufficient activation of NF-B to direct cytokine expression
and generate an intense neutrophilic lung inflammation. This response
is abrogated by coadministration of adenoviral vectors expressing a
dominant inhibitor of NF-B, which indicates that the inflammatory
state resulting from lung epithelial expression of cIKK1 and cIKK2 is
specifically related to activation of the NF-B pathway. Our findings
suggest an important role for epithelial cell NF-B activation in
regulating the generation of neutrophilic lung inflammation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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For construction of replication-deficient adenoviral vectors
that express activators of NF-B, we obtained cIKK1 and cIKK2 from
Dr. F. Mercurio (Signal Pharmaceutical, San Diego, CA). IKK1 and IKK2
were made constitutively active by Ser-Glu mutations in critical serine
residues that are phosphorylated in the active kinase
(Ser176, Ser180 in IKK1 and
Ser177, Ser181 in IKK2)
(12). The replication-deficient recombinant adenovirus
(Ad) type 5 was used. An expression cassette containing a CMV promoter
driving the expression of cIKK1 or cIKK2 was inserted into this vector.
Adenoviral vectors expressing a dominant inhibitor of NF-B
(IB-DN), which represents a S36–40A mutant of the avian
IB- that cannot be phosphorylated or degraded, and -gal have
been previously reported. Adenoviral vectors expressing
luciferase were a gift from Dr. A. Powers (Vanderbilt University,
Nashville, TN). Adenoviral vectors were propagated, purified, and
stored at -70°C.
Animal model
Transgenic mice expressing Photinus luciferase cDNA under control of the proximal 5' HIV-long terminal repeat (LTR) mice on a C57B6/DBA background weighing 20–30 g were used (24). Mice were treated with IT administration of adenoviral vectors after sedation with ketamine/xylazine. Mouse tracheas were directly exposed by surgical resection, pierced with a 26-gauge needle, and injected with 100 µl of the Ad preparation diluted in sterile PBS. The neck wound was closed with sterile sutures under aseptic conditions.
Mice were asphyxiated with CO2 and lungs were removed. One lung was ground in 1 ml of reporter lysis buffer (Promega, Madison, WI) and stored at -20°C for luciferase assays and the other lung was frozen at -70°C. In some experiments, tracheas were cannulated and lungs were lavaged in situ with sterile pyrogen-free physiological saline that was instilled in four 1-ml aliquots and gently withdrawn with a 1-ml tuberculin syringe.
In vivo measurement of luciferase gene expression by bioluminescence
Mice were anesthetized and shaved over the chest and abdomen before imaging. Luciferin (1 mg/mouse in 200 µl of isotonic saline) was administered by i.p. injection, and mice were imaged with an intensified charge- coupled device (ICCD) camera (model C2400-32; Hamamatsu, Bridgewater, NJ) as previously reported (25, 26). This system consists of an image intensifier coupled to an 8-bit charge-coupled device camera, allowing for 256 intensity levels for each pixel. For the duration of photon counting, mice were placed inside a light tight box that also houses the camera. Light emission from the mouse was detected as photon counts using the ICCD camera and customized image processing hardware and software (Hamamatsu). The imaging duration (3 min) was selected to avoid saturating the camera during image acquisition. Quantitative analysis was accomplished by 1) defining a standard area (region of interest) in the 8-bit intensity image corresponding to the region of the chest overlying the mid lung zone and 2) determination of total integrated photon intensity over the region of interest. Photon counts were obtained before and after treatment with Ad so that each mouse could be used as its own control. For presentation, a 4-bit (16 intensity levels) digital false-color photon emission image was generated for each captured image according to the same false-color scale. To visualize the dimmer parts of the image, the brighter pixels in the images are displayed as white (thus appearing saturated); however, detected light emission for each image was well below the saturation limit for the camera.
Measurement of luciferase activity in lung tissue
Luciferase activity was measured in postmortem tissue samples by adding 100 µl of freshly reconstituted luciferase assay buffer (Promega) to 20 µl of lung tissue homogenate. Luciferase activity was measured in a Monolight 3010 Luminometer (Analytical Luminescence Laboratory, San Diego, CA) and expressed as relative light units (RLU) normalized for protein content, which was measured according to the Bradford assay (27).
Lung lavage total and differential cell counts
Lung lavage fluid was centrifuged at 400 x g for 10 min to separate cells from supernatant. Supernatant was saved separately and frozen at -70°C. The cell pellet was suspended in serum-free RPMI 1640 culture medium, and total cell counts were determined on a grid hemocytometer. Differential cell counts were determined by staining cytocentrifuge slides with a modified Wright stain (Diff-Quick; Baxter, McGraw Park, IL) and counting 400–600 cells in complete cross-sections.
Macrophage-inflammatory protein (MIP) 2 and KC ELISA
MIP-2 and KC levels were measured using a sandwich ELISA according to the manufacturer’s instructions (R&D Systems, Minneapolis, MN).
Ribonuclease protection assay (RPA)
RPA employing both murine cytokine and chemokine templates were
done with the RiboQuant multiprobe RPA system (BD PharMingen, San
Diego, CA) according to the manufacturer’s directions. Briefly, the
probes were radiolabeled by adding [-32P]UTP
to a reaction mixture that included template, transcription buffer, and
T7 polymerase. After incubation, DNase was added and RNA probes were
purified by phenol-chloroform extraction followed by ethanol
precipitation. The probes were resuspended in hybridization buffer and
diluted to 4 x 105 counts/min per µl.
Twenty micrograms of total RNA from the lung was dried in a vacuum
evaporator centrifuge and resuspended in 8 µl of hybridization
buffer. Two micrograms of the labeled probe was added to each sample,
heated to 90°C, and incubated overnight at 56°C. RNase was added,
followed by proteinase K, and the resultant digest was
phenol-chloroform extracted and precipitated with ethanol. Loading dye
was added to the dried pellets, the samples were heated briefly to
90°C, and a 5% polyacrylamide gel was run, dried, and subjected to
autoradiography.
Western blot
Twenty-five micrograms of protein from tissue homogenates or
lung lavage cells was separated on a 10% acrylamide gel, transblotted,
and immunodetected. cIKK1 was detected with Abs to a hemagglutinin tag
present on the protein (Babco, Richmond, CA). cIKK2 was detected with
Abs to a FLAG tag present on the protein
(anti-FLAGM2 mAb; Sigma-Aldrich, St. Louis,
MO). IB-DN was detected with specific antiserum that does not
cross-react with native murine IB- or (24). Abs
to RelA were obtained from Santa Cruz Biotechnology (Santa Cruz,
CA).
-gal detection
Frozen sections of lung tissue were cut, fixed in 50%
glutaraldehyde in PBS for 20 min, and stained with
5-bromo-4-chloro-3-indolyl -D-galactoside (X-gal)
solution (5 mM potassium ferrocyanide, 5 mM potassium ferrocyanide, 2
mM MgCL2, and 1 mg/ml 20% X-gal) for 12–18
h.
EMSA
After preparation of nuclear extracts, EMSA for NF-B binding
activity were done as previously described (28). A
double-stranded oligonucleotide probe containing consensus NF-B
motif (Stratagene, La Jolla, CA) was used in these studies.
Lung histology
Lungs were removed en bloc after tracheal ligation, preserved in 10% formalin, and subsequently embedded in paraffin. H&E or Wright-Giemsa stain was then performed on 5-µm sections.
Statistical analysis
For comparison among groups, a one-way ANOVA was used with the Tukey-Kramer multiple comparisons test (p <0.05 were considered to be significant).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We performed initial experiments with adenoviral vectors
expressing luciferase (Ad-luc) and -gal (Ad--gal) reporters to
determine the timing and localization of transgene expression after IT
injection. Mice were treated with Ad-luc at a dose of 3 x
109 PFU diluted in 100 µl of PBS (Fig. 1). Control mice were treated with 100
µl of sterile PBS by IT injection. To detect luciferase activity in
vivo, wild-type C57B/6 mice were treated with 1 mg of luciferin by i.p.
injection and imaged with an ICCD camera at 24, 72, and 96 h after
administration of adenoviral vectors. Untreated mice and control mice
treated with PBS showed no detectable bioluminescence, but mice treated
with Ad-luc demonstrated luciferase activity (as detected by photon
emission) from the chest by 24 h and this bioluminescence
persisted through 96 h, which was the last time point evaluated
(Fig. 1A). Of note, bioluminescence was not detected in any
organ outside the chest following IT injection of adenoviral vectors.
At 96 h, lungs were removed after i.p. luciferin injection and
placed in a culture dish with RPMI 1640 medium. Fig. 1B
shows ex vivo bioluminescence of lungs from three mice treated with
Ad-luc. Bioluminescence was not detected from liver, spleen, or kidneys
of these mice ex vivo and no bioluminescence was detected from any
organ of mice treated with PBS alone (data not shown). These studies
clearly demonstrate transgene expression primarily in the lungs that
persists for at least 96 h after IT administration of adenoviral
vectors.
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-gal at 3 x 109 PFU. Ninety-six
hours after administration of Ad--gal, mice were harvested and X-gal
staining was performed on frozen lung sections (Fig. 1C).
Mice treated with Ad--gal showed staining almost exclusively in
large airway epithelium. Staining was not detected on similarly
prepared lung sections from PBS-treated controls (data not shown).
These findings show that adenoviral vectors can be used to deliver
transgenes selectively to airway epithelium.
Because of reports that high doses of adenoviral vectors can
cause lung inflammation and that adenoviral vectors alone can activate
NF-B in cultured lung epithelial cell lines (23, 29, 30, 31), we performed experiments to define a titer of
adenoviral vectors that results in transgene expression in the lungs in
vivo without substantial nonspecific activation of NF-B or other
signs of inflammation. To accomplish this, we used a line of transgenic
NF-B reporter mice that possesses a portion of the proximal 5' human
HIV-LTR driving the expression of luciferase ((referred to as HLL mice
(HIV-LTR/luciferase)) (24). The proximal HIV-LTR is a
well-characterized NF-B responsive promoter containing a TATA box,
an enhancer region between -82 and -103 with two NF-B motifs, and
three Sp1 boxes from -46 to 78. In cell culture, NF-B activation is
absolutely required for transcriptional activity of the HIV-LTR. We
have shown that luciferase activity in these transgenic mice reflects
NF-B activation over time (24). We have used
bioluminescence imaging after luciferin injection to measure luciferase
activity in these animals and have shown that this methodology
correlates well with luciferase activity measured in a variety of
tissues (25, 26). Using HLL mice, we performed
dose-ranging experiments using
107–1011 PFU of Ad-
-gal delivered by IT injection. HLL mice were imaged after i.p.
luciferin (1 mg) at baseline and at 24, 72, and 96 h after
adenoviral administration. At doses of 1010 PFU
and higher, increased bioluminescence from the lungs was detected (data
not shown). Subsequent studies were done with 3 x
109 or lower PFU, doses that did not result in
increased bioluminescent detection of luciferase activity in HLL mice
at the time points evaluated.
At the doses of adenoviral vectors that did not result in
nonspecific activation of NF-B or vector-induced lung inflammation,
we were able to identify specific transgene products expressed in lung
tissue by Western blot (Fig. 2). Mice
were treated with adenoviral vectors expressing 1) cIKK1 or cIKK2
(Ad-cIKK1 and Ad-cIKK2, respectively) at 3 x
108 PFU, 2) a dominant inhibitor of NF-B,
which is a S36/40A mutant of avian IB-, designated as IB-DN
(Ad-IB-DN) at 3 x 109 PFU, or 3)
Ad--gal at 3 x 109 PFU. Mice treated
with Ad-cIKK1, Ad-cIKK2, or Ad-IB-DN demonstrated expression of
cIKK1, cIKK2, or IB-DN in a corresponding fashion that was
detectable in lungs harvested 96 h after IT injection of the
specific adenoviral construct. These proteins were not detectable in
homogenates from control mice treated with Ad--gal (Fig. 2). To
determine whether transgene expression was limited to the epithelium,
we performed separate experiments in which we evaluated transgene
expression in lung lavage cells 96 h after IT injection of
adenoviral vectors (Fig. 3). As shown,
Ad-cIKK1 and Ad-cIKK2 expression was not detectable in lung lavage
cells. Therefore, in these experiments, we were able to identify doses
of adenoviral vectors that resulted in epithelial transgene expression
without significant nontransgene-related activation of NF-B.
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B activation
in vivo in a transgene-specific manner
We investigated whether IT administration of Ad-cIKK1 could induce
NF-B activation and luciferase production in the lungs of HLL
reporter mice. In these studies, mice were imaged by bioluminescence
immediately before treatment (baseline) and at 24, 72, and 96 h
after IT injection of adenoviral vectors. Luciferin was administered by
i.p. injection (1 mg in 200 µl of PBS) 30 min before bioluminescence
imaging and photonic counts were measured over a standardized area of
the chest overlying lung tissue. Mice treated with Ad-cIKK1 showed a
significant time-dependent increase in photonic counts, with peak
activation at 72 h (5153 ± 127 photonic counts for Ad-cIKK1
compared with 1698 ± 667 photonic counts for Ad--gal controls,
p < 0.05, n = 5; Fig. 4). Mice were sacrificed at 96 h and
the final photonic counts were compared with luciferase activity
measured in lung homogenates. Luciferase activity in lung tissue
homogenates of mice treated with Ad-cIKK1 was much higher than in lung
homogenates from mice treated with Ad--gal (238 ± 44 RLU/µg
protein for cIKK1 group vs 134 ± 33 RLU/µg protein for -gal
group (p < 0.05)) and there was a close
correlation between bioluminescent emission from the lungs at 96 h
and luciferase activity in lung homogenates
(R2 = 0.88, p <
0.001; data not shown). In untreated mice, luciferase activity in lung
homogenates was 110 ± 20 RLU/µg protein. These data indicate
that treatment with Ad-cIKK1 activates NF-B-dependent luciferase
activity in lung tissue of HLL mice.
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B by cIKK1, we performed experiments
using adenoviral vectors that express a transdominant inhibitor of
NF-B (IB-DN). Since this IB- mutant cannot be
phosphorylated and degraded, NF-B is sequestered in the cytoplasm
and cannot be activated by IKK. We treated HLL mice with Ad-IB-DN
or Ad-cIKK1 alone or the combination of Ad-cIKK1 plus Ad-IB-DN.
In these experiments, Ad-cIKK1 was administered at a dose of 3 x
108 PFU and Ad-IB-DN was given at 3 x
109 PFU. Bioluminescent imaging of HLL mice
was done at 24, 72, and 96 h after IT administration of
adenoviral vectors. At 96 h, lungs were harvested for luciferase
activity measurements and measurement of NF-B activation.
Bioluminescent imaging of mice at 72 h after treatment is shown in
Fig. 5A. Baseline imaging of a
mouse is shown as well as images of mice treated with Ad-IB-DN,
Ad-cIKK1, and Ad-cIKK1 plus Ad-IB-DN. In these pseudocolor images
in which white represents the greatest intensity of photon emission,
minimal light emission is detected over the lungs (arrows) at baseline
and after treatment with Ad-IB-DN. After Ad-cIKK1, light emission
from the thorax is significantly increased; however, coadministration
of Ad-IB-DN with Ad-cIKK1 completely abolishes the increase in
light emission induced by cIKK1 expression. Fig. 5B
summarizes detected photon emission from the lungs in these experiments
at 72 h after IT injection. Mice treated with Ad-IB-DN did
not show a significant increase in photon emission over baseline,
whereas mice treated with Ad-cIKK1 alone had markedly increased
photonic counts from the lungs (1800 ± 387 for Ad-IB-DN
group vs 6100 ± 900 for Ad-cIKK1 group, p <
0.001). This increase in photon emission with Ad-cIKK1 treatment was
completely blocked by the coadministration of a 10-fold excess of
Ad-IB-DN (2000 ± 219). Similar differences among groups
were found at 24 and 96 h after adenoviral vector treatment.
Measurements of bioluminescence from the thorax correlated closely with
postmortem measurements of luciferase activity in lung tissue from mice
harvested 96 h after adenoviral vector administration (Fig. 5C). Treatment with Ad-cIKK1 resulted in increased lung
luciferase activity (normalized for total protein content) compared
withtreatment with Ad-IB-DN alone, and this induction of
luciferase activity by cIKK1 was completely blocked by coadministration
of Ad-cIKK1 plus Ad-IB-DN.
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B in the lungs in these
experiments, we measured immunoreactive RelA in lung nuclear protein
extracts by Western blot. Fig. 5D demonstrates the presence
of RelA protein in nuclear extracts from three mice that were treated
with Ad-cIKK1 and the absence of detectable RelA in extracts from four
mice treated with either Ad-IB-DN alone or the combination of
Ad-cIKK1 plus Ad-IB-DN. These data indicate that
IB-DN prevented nuclear localization of RelA in response to
treatment with the Ad-cIKK1. Also, the presence of nuclear RelA
correlated with lung expression of luciferase in these experiments. In addition to adenoviral vectors expressing cIKK1, we constructed adenoviral vectors expressing cIKK2. We administered Ad-cIKK2 or Ad-cIKK1 alone (3 x 109 PFU) or in combination (1.5 x 109 PFU of each) and evaluated lung bioluminescence at 24, 72, and 96 h. In this experiment, photon emission was highest in the group that received Ad-cIKK2 and intermediate in the group that received combined cIKK1 plus cIKK2. Fig. 6A shows detected photon emission from the lungs at 72 h after adenoviral vector administration. Mean photonic counts from the lungs of mice treated with Ad-cIKK2 were 18,340 ± 3,257 compared with 15,750 ± 1,780 photonic counts in the combined Ad-cIKK1 and Ad-cIKK2 group and 7,678 ± 789 in the Ad-cIKK1 group. Luciferase activity measurements in lung homogenates at 96 h were also highest in the group that received Ad-cIKK2 (Fig. 6B). Although both Ad-cIKK1 and Ad-cIKK2 induced luciferase activity in the lungs of HLL reporter mice, cIKK2 induced higher levels of luciferase in the lungs, and there was no synergistic effect of adding both vectors together.
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B-DN with Ad-cIKK2 blocks NF-B activity and
NF-B-dependent luciferase activity in the lungs of HLL reporter
mice. NF-B activation was evaluated by EMSA from lung nuclear
protein extracts 96 h after adenoviral vector treatment (Fig. 7). Bands on EMSA were identified by cold
and nonspecific competition and Ab supershifts as containing RelA/p50
heterodimers and p50 homodimers as indicated. As shown, lung nuclear
protein extracts from mice treated with Ad-cIKK1 (Fig. 7, lanes 3 and 4) or Ad-cIKK2 (lanes
7 and 8) demonstrated intense binding to a consensus
NF-B oligonucleotide, whereas nuclear protein extracts from mice
treated with Ad-IB-DN in combination with Ad-cIKK1 or Ad-cIKK2
had minimal binding of the RelA/p50 heterodimer band
(lanes 5 and 6 and 9 and
10, respectively). Induction of lung luciferase activity by
Ad-cIKK2 in these experiments was also blocked by coadministration of
Ad-IB-DN (data not shown). These data clearly indicate that
NF-B activation in lungs results from treatment with adenoviral
vectors that express cIKK1 or cIKK2 and that this activation can be
specifically inhibited by expression of IB-DN.
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B in the lungs results in increased cytokine
expression and neutrophilic lung inflammation
To evaluate the effects of NF-B activation in lung epithelium,
we used multiprobe RPA from total lung RNA to determine cytokine and
chemokine gene expression 96 h after treatment with Ad-IB-DN
alone, Ad-cIKK1, Ad-cIKK2, or a combination of Ad-cIKK1 (or Ad-cIKK2)
plus Ad-IB-DN. In HLL mice that were treated with Ad-cIKK1
or Ad-cIKK2 alone, mRNA expression of a variety NF-B-dependent
chemokines was induced, including RANTES, eotaxin, MIP-1, MIP-1,
MIP-2, IFN-inducible protein 10, and monocyte chemoattractant
protein 1 (Fig. 8A). Increased
expression of all of these chemokines was blocked by combined treatment
with Ad-cIKK1 (or cIKK2) and Ad-IB-DN. Expression of mRNA for
cytokines TNF- and IL-6 was also found in the lungs of mice treated
with Ad-cIKK1 or Ad-cIKK2, whereas in mice that received Ad-IB-DN
in addition to Ad-cIKK1 or Ad-cIKK2, mRNA for these cytokines was only
minimally detectable (Fig. 8B).
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B-dependent chemokines are thought to be important for
neutrophil recruitment (1). In untreated mice, no
MIP-2 or KC was detectable in lung lavage fluid and in mice
treated with Ad--gal small amounts of both chemokines were
present (Fig. 9A). However,
mice treated with Ad-cIKK1 or Ad-cIKK2 had significant elevation of
lung lavage levels of these chemokines compared with mice treated with
Ad--gal. Chemokine levels of mice coadministered Ad-cIKK1 plus
Ad-IB-DN or Ad-cIKK2 plus Ad-IB-DN were similar to those
seen after treatment with Ad--gal.
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B activation in lung epithelium results
in recruitment of neutrophils to lungs or otherwise alters lung
histology. Fig. 9B shows cell counts from lung lavage
obtained 96 h after adenoviral vector administration. In these
experiments, mice treated with Ad-cIKK1 or Ad-cIKK2 had abundant
numbers of neutrophils that could be recovered in lung lavage
compared with mice that were treated with Ad--gal.
Simultaneous administration of Ad-IB-DN with Ad-cIKK1 or Ad-cIKK2
abolished the increase in neutrophil counts induced by expression of
cIKK1 or cIKK2 alone. In untreated mice, lung lavage neutrophil counts
were very low (<1 x 104 cells) and similar
to the group treated with Ad-IB-DN alone (data not shown).
Significant numbers of lymphocytes or eosinophils were not identified
in the lung lavage in any of the treatment groups.
We examined lung histology of mice 96 h after treatment with
adenoviral vectors to assess the extent of inflammatory changes. Lung
tissue from mice that were treated with either Ad-cIKK1 or Ad-cIKK2
showed pronounced inflammatory changes that included intra-alveolar
accumulation of inflammatory cells, predominantly neutrophils. Alveolar
septal thickening was also observed in lungs of these mice. Fig. 10 shows representative lung histology
from a mouse treated with Ad-cIKK2 (similar lung inflammation was found
after treatment with Ad-cIKK1). No histological abnormalities were
identified in mice treated with Ad--gal (Fig. 10) or Ad-IB-DN.
As with other parameters of lung inflammation, coadministration of
Ad-IB-DN with Ad-cIKK1 or cIKK2 prevented the histological
changes seen in mice treated with Ad-cIKK1 or Ad-cIKK2 alone (data not
shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B reporter mice and
adenoviral vectors to mediate activation and inhibition of NF-B
specifically in lung epithelium. This approach employs novel
methodology to study this signal transduction pathway in live animals.
Expression of constitutively active forms of IKK1or IKK2 in airway
epithelium activates NF-B with resultant cytokine and chemokine
production and neutrophilic lung inflammation. This inflammatory
response is blocked by a dominant inhibitor of NF-B, showing that
generation of inflammation by Ad-cIKK1 and Ad-cIKK2 is specifically
dependent on NF-B. Together, these data show that activation of
NF-B in airway epithelium by cIKK1 or cIKK2 is sufficient to produce
neutrophilic lung inflammation.
Recombinant adenoviral vectors are efficient vehicles for delivery of
genes and are useful for targeting airway epithelium. Ad offer the
advantage of high titer, stability, and the ability to achieve gene
transfer independent of the cell replication status. Several recent
studies have used adenoviral vectors to manipulate NF-B activation
to study the impact of this pathway on inflammation. For example,
expression of wild-type IKK2 was shown to induce synovial inflammation
after intra-articular gene transfer in rats, and this inflammation was
inhibited by expression of a dominant negative form of IB-
(32). Another group found that expression of
adenoviral-delivered RelA in the pancreas resulted in pancreatitis
(33) and coadministration of Ad-IB- diminished the
inflammatory response. In addition, several groups, including ours,
have used adenoviral vectors expressing a dominant inhibitor of the
NF-B pathway to block NF-B in target cells and tissues to assess
the impact on NF-B-mediated inflammation (24, 34, 35).
These studies show that adenoviral vector-mediated gene delivery can be
used to investigate the NF-B pathway; however, there are issues that
can limit the utility of this approach. At high doses,
replication-deficient adenoviral vectors have been shown to cause a
significant inflammatory response (29, 30, 31). This
inflammatory response has been described to consist of an early phase
(within 24 h) characterized by the accumulation of neutrophils and
macrophages, followed by a later phase that develops after 5 days,
consisting primarily of lymphocytes (29, 30, 31). In our
studies, we have shown that a dose of replication-deficient Ad could be
selected to achieve high-level transgene expression in airway
epithelium with minimal viral vector-induced inflammation. In contrast,
the inflammatory response induced by expression of Ad-cIKK1 or Ad-cIKK2
was significantly greater than control Ad and was specifically
inhibited by expressing a transdominant NF-B inhibitor. By carefully
controlling these experiments, we have been able to show that
adenoviral vector-mediated expression of cIKK1 or cIKK2 results in lung
inflammation through activation of NF-B by these kinases.
The differential roles of the two IKKs are not completely understood;
however, IKK2 is thought to be the principal kinase involved in
induction of NF-B by inflammatory stimuli such as TNF- and
IL-1 (15, 16, 17, 36). Our study shows that a
constitutively active mutant form of either IKK is capable of
activating NF-B in vivo and inducing a NF-B-dependent
inflammatory response. In our studies, treatment with Ad-cIKK2 induced
higher levels of NF-B-dependent luciferase in the lungs of HLL
reporter mice than treatment with Ad-cIKK1. Although it is possible
that expression of cIKK2 resulted in more efficient activation of
NF-B than cIKK1, we cannot exclude the possibility that differences
in NF-B activation in these studies resulted from disparity in the
level of expression of cIKK1 and cIKK2. Regardless, the major finding
in these studies is that both kinases are competent to cause NF-B
activation and inflammation in vivo.
The specific functions of the NF-B activation pathway in various
lung cell types are not well defined. Although airway epithelial cells
in culture have been shown to activate NF-B and produce chemokines
in response to various stimuli, the role of these cells in regulation
of acute neutrophilic inflammation has not been specifically evaluated.
In this study, we show that bronchial epithelial cells are fully
capable of inducing an inflammatory response mediated through NF-B
activation and it is possible that targeting airway epithelial cells
for anti-inflammatory therapy would be beneficial for treatment of
inflammatory lung diseases.
In summary, we have shown that airway delivery of adenoviral vectors
can be used to study signal transduction pathways in lung epithelial
cells. Using this strategy, we have demonstrated that selective
activation of the NF-B transcription factor complex in lung
epithelium by expression of cIKKs is sufficient to induce
cytokine/chemokine gene expression and to generate neutrophilic lung
inflammation. We believe that therapies directed toward inhibition of
NF-B in specific lung cell types could be beneficial for treatment
of a variety of inflammatory lung diseases.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Timothy S. Blackwell, Allergy, Pulmonary and Critical Care Medicine, Vanderbilt University School of Medicine, T-1217 MCN, Nashville, TN 37232-2650. E-mail address: timothy.blackwell{at}mcmail.vanderbilt.edu
3 Abbreviations used in this paper: IKK, IB kinase; Ad, adenovirus; Ad-luc, Ad-luciferase; -gal, -galactosidase; IT, intratracheal; cIKK, constitutively active IKK; ICCD, intensified charge-coupled device; MIP, macrophage-inflammatory protein; RPA, ribonuclease protection assay; X-gal, 5-bromo-4-chloro-3-indolyl -D-galactoside; LTR, long terminal repeat; RLU, relative light units; HLL, HIV-LTR/luciferase.
Received for publication August 5, 2002. Accepted for publication November 11, 2002.
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