Division of Allergy, Pulmonary, and Critical Care Medicine, Department of Medicine, Vanderbilt University School of Medicine, Nashville 37232-2650; and Department of Medicine, Department of Veterans Affairs, Nashville, Tennessee 37212
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ABSTRACT |
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We examined the relationship between nuclear
factor (NF)-B DNA binding activity, cytokine gene
expression, and neutrophilic alveolitis in rats after intratracheal
(IT) instillation of endotoxin [lipopolysaccharide (LPS)].
NF-
B activation in lung tissue mirrored neutrophilic alveolitis
after IT LPS instillation, with NF-
B activation and neutrophilic
influx beginning 2 h after IT LPS doses of 0.01 mg/kg or greater. In
lung lavage fluid cells, however, transient NF-
B
activation was present in alveolar macrophages by 15 min after IT LPS
instillation, followed by a second peak of NF-
B activation
corresponding to the onset on neutrophilic alveolitis. For cytokines
thought to be NF-
B dependent, two different patterns of mRNA
expression were found. Interleukin (IL)-1
, IL-1
, and tumor
necrosis factor-
showed increased mRNA by 30 min after IT LPS
instillation, but IL-6- and cytokine-induced neutrophil chemoattractant
mRNAs were not substantially increased until 2 h after IT LPS
instillation. Therefore, IT LPS causes differential NF-
B activation
in air space cells and lung tissue, which likely determines production
of key cytokines and directs the evolution of neutrophilic alveolitis.
nuclear factor-B; lung; neutrophil; inflammation; chemokine; macrophage
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INTRODUCTION |
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PRODUCTION OF ACUTE inflammatory cytokines and influx
of activated neutrophils into the lungs are thought to be important in
the pathogenesis of lung injury in acute respiratory
distress syndrome, which can occur as the result of direct pulmonary
insults including bacterial pneumonia, gastric acid aspiration, and
inhalation of toxic gases. In animal models, Ulich et al. (20) and
other investigators (8, 11, 27) have shown that intratracheal (IT)
administration of bacterial endotoxin [lipopolysaccharide (LPS)] causes both lung cytokine production and neutrophilic
influx. In this study, we examined the relationship between cytokine
gene expression, neutrophilic alveolitis, and activation of the
transcription factor complex nuclear factor (NF)-B in rats treated
with IT LPS.
NF-B is a ubiquitous transcription factor that exists as a dimer of
two Rel proteins. In unstimulated cells, NF-
B is bound to an
inhibitor (I
B) that anchors the protein complex in the cell
cytoplasm. Inflammatory stimuli, including LPS, tumor necrosis factor
(TNF)-
, interleukin (IL)-1, viral proteins, and mitogens, cause
phosphorylation, ubiquitinization, and degradation of I
B. Liberated
NF-
B then translocates to the nucleus, binds to specific sites on
DNA, and enhances the expression of a variety of genes that are
important for promoting acute inflammation (4, 13). Mediators of
inflammation that are transcriptionally regulated by NF-
B in vitro
include cytokines (TNF-
, IL-1
, and IL-6), C-X-C chemokines
[IL-8; GRO
, -
, and -
; macrophage inflammatory protein-2;
and cytokine-induced neutrophil chemoattractant (CINC)], and
enzymes (cyclooxygenase-2 and inducible nitric oxide synthase) (4, 13).
Our hypothesis in these studies was that IT LPS would induce specific
patterns of NF-B activation in lung lavage fluid cells and lung
tissue and that NF-
B activation would correlate with NF-
B-dependent cytokine gene expression and neutrophilic alveolitis. We anticipated that the earliest event in the development of
neutrophilic lung inflammation would be increased nuclear binding
activity of NF-
B proteins in alveolar macrophages because these
cells are thought to play a role in the initiation of the inflammatory cascade. We evaluated the dose requirements of LPS and the timing of
NF-
B activation in whole lung tissue and lung lavage fluid cells and
found a differential pattern of NF-
B activation. We compared the
pattern of NF-
B binding activity to neutrophilic alveolitis, gene
expression of a panel of acute inflammatory cytokines, and the rat
neutrophil chemoattractant chemokine CINC. Our findings show that
NF-
B activation in the lung after IT LPS instillation correlates
with cytokine mRNA expression and neutrophilic alveolitis, supporting
the idea that NF-
B activation is a pivotal event in the generation
of neutrophilic lung inflammation. Interestingly, a burst of NF-
B
binding activity in alveolar macrophages precedes neutrophilic
inflammation and correlates with whole lung gene expression of TNF-
and IL-1, consistent with the hypothesis that alveolar macrophage
activation triggers the inflammatory process.
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METHODS |
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Animal model. Male Sprague-Dawley rats
weighing between 200 and 300 g were used in all experiments.
Escherichia coli LPS (serotype 055:B5;
Sigma, St. Louis, MO) was given by a single IT injection in doses of
0-6 mg/kg. For IT injections, the rats were anesthetized with
ketamine and xylazine, and LPS (diluted in sterile physiological saline) was instilled directly into the trachea in a total volume of
300-400 µl. The rats were asphyxiated with carbon dioxide, the
tracheae were cannulated after death, and the lungs were lavaged in
situ with sterile pyrogen-free physiological saline. Saline was
instilled in two 5-ml aliquots and gently withdrawn with a 5-ml
syringe. For other studies, the lungs were removed, quickly frozen in
liquid nitrogen, and stored at 70°C.
Cell counts and differentials. Lung lavage fluid was centrifuged at 500 g for 10 min to separate cells from supernatant. Pelleted cells were resuspended in a small amount of physiological saline with 1% BSA. Total cell counts were determined on a grid hemacytometer. Differential cell counts were enumerated on cytocentrifuge slides that were stained with a modified Wright stain (Diff-Quik) by counting 400-600 cells in cross section.
Nuclear protein extractions for lavage fluid cells and lung tissue. Nuclear proteins from lavage fluid cells and from tissue were prepared as previously described (1, 2). For lavage fluid cell nuclear extracts, lavage fluid cells from two different rats at each time point were combined to ensure adequate nuclear protein yields.
Electrophoretic mobility shift assays.
An oligonucleotide probe containing a consensus NF-B motif,
5'-GATCGAGGGGACTTTCCCTAGC-3' (Stratagene, La Jolla, CA),
was used in these studies. End labeling was accomplished by treatment
with T4 kinase in the presence of [32P]ATP. Labeled
oligonucleotide was column purified on a Sephadex G-25M column
(Pharmacia Biotech, Piscataway, NJ). Labeled double-stranded probe
(~100,000 counts/min) was added to 5 µg of nuclear protein in the
presence of binding buffer (Stratagene). This mixture was incubated at
25°C for 20 min and separated by electrophoresis on a 6%
polyacrylamide gel in 1× Tris-borate-EDTA buffer. The gels were
vacuum-dried and subjected to autoradiography. Cold competition was
done by adding 50 ng of specific unlabeled double-stranded probe to the
reaction mixture. The specificity of the DNA binding was confirmed by
adding an excess of an unrelated nonspecific DNA sequence,
5'-GATCGAATGCAAATCACTAGCT-3', that contains an Oct-1 binding motif. On some gels, supershift assays were done with polyclonal antibodies to Rel A p65 obtained from Santa Cruz
Biotechnology. Two micrograms of the antibody were added
to the reaction mixture, and the samples were incubated at 25°C for
1 h before gel loading.
Tissue RNA extractions. Total RNA was purified with a modification of the method of Chirgwin et al. (6). Frozen lung tissue was mixed with 1 ml of TRI REAGENT (Molecular Research Center, Cincinnati, OH) and ground in a tissue homogenizer. The samples were transferred to 1.5-ml Eppendorf tubes, and RNA was extracted with phenol-chloroform and precipitated with isopropanol. The RNA pellet was then washed with 75% ethanol, air-dried, and dissolved in 50-100 µl of 30% formamide-10% formaldehyde. Total RNA was quantitated by determining the light absorbance at 260 nm.
Northern blots. Total RNA was separated by electrophoresis on a 1% agarose-formaldehyde gel, transblotted to nitrocellulose, and fixed with ultraviolet light (Stratagene 1800). The filters were prehybridized, then hybridized with a specific 32P end-labeled 30-base oligonucleotide probe for CINC with the sequence 5'-GCGGCATCACCTTCAAACTCTGGATGTTCT-3', which is complementary to nucleotides 134-164 of CINC cDNA. Blots were high-stringency washed, and CINC mRNA was detected by autoradiography in the presence of intensifying screens.
RNase protection assay. RNase
protection assays (RPAs) were done with the RiboQuant multiprobe RPA
system (PharMingen, San Diego, CA). A rat cytokine template set (rCK-1)
was used. The probes were made and radiolabeled by adding
[-32P]UTP to a
reaction mixture including template, transcription buffer, and T7
polymerase. After incubation, DNase was added, and the RNA probes were
purified by phenol-chloroform extraction followed by ethanol
precipitation. The probes were resuspended in hybridization buffer and
diluted to 4 × 105
counts · min
1 · µl
1.
Twenty micrograms of total RNA from the lung were dried in a vacuum
evaporator centrifuge and resuspended in 8 µl of hybridization buffer. Two micrograms of labeled probe were added to each sample, which was then heated to 90°C and incubated overnight at 56°C. RNase was then added, followed by proteinase K. Resultant RNase digests
were then phenol-chloroform extracted and precipitated with ethanol.
Loading dye was added to the dried pellets, and the samples were heated
briefly to 90°C. A 5% polyacrylamide gel was then run, dried, and
subjected to autoradiography.
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RESULTS |
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Initially, we determined the time course and dose response for IT LPS
induction of neutrophilic alveolitis and NF-B binding activity in
nuclear extracts of lung tissue (Figs. 1 and 2). Figure 1A shows
the percentage of alveolar macrophages and neutrophils in lung lavage
fluid 0, 0.5, 1, 2, 4, and 6 h after treatment with IT LPS at 6.0 mg/kg, a dose that causes neutrophilic lung inflammation
after intraperitoneal injection of LPS (5). In control lungs and up to
1 h after IT LPS instillation, >96% of lavage fluid cells were
alveolar macrophages. By 2 h, neutrophils accounted for 65% of lavage
fluid cells, and this percentage increased to 82% by 4 h and to 94.5%
by 6 h after IT LPS instillation. The total number of lavage fluid
neutrophils and macrophages is reported in Fig.
1B. As shown, there was a fivefold
rise in the total number of lavage fluid cells by 6 h, which was
entirely due to a massive increase in the number of neutrophils.
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Using lung tissue from these rats, we next determined the effect of IT
LPS on NF-B binding activity. Figure
1C shows the time course for the
appearance of NF-
B binding activity in nuclear protein extracts from
lung tissue. With electrophoretic mobility shift assays from rat lung
tissue, we can detect up to three bands, which we labeled
bands A,
B, and
C. These bands have been identified by
supershifted electrophoretic mobility shift assays with antibodies to
the NH2 terminus and COOH terminus
of Rel A p65 and to p50 (1, 2) (data not shown). Band
A contains intact Rel A/p50 heterodimers (binds to all
three antibodies), band B contains p50
homodimers (binds to only the p50 antibody), and band
C contains Rel A/p50 heterodimers in which the Rel A is
truncated at the COOH terminus by an ex vivo event during nuclear
protein extraction [binds to p50 and the
NH2-terminal Rel A antibody but
not to the COOH-terminal Rel A antibody (1, 2); therefore,
bands A and
C contain "classic" NF-
B (Rel
A/p50)]. Bands containing Rel A are of particular interest
because Rel A, but not p50, contains a COOH-terminal transcription
activation domain (12). In Figure 1C,
only bands B and
C were identified. Lung Rel A/p50
(band C) binding activity was
minimal at 30 min and 1 h after IT LPS instillation but was markedly
upregulated by 2 h after treatment with IT LPS and sustained to 6 h.
The p50 homodimer (band B) was
present in untreated rats and remained detectable throughout the time
course. Therefore, in lung tissue, NF-
B (Rel A/p50) binding activity correlates directly with neutrophil influx into the alveolar space, which implies that the detected NF-
B binding activity could be due,
at least in part, to NF-
B activation in the neutrophils themselves.
After completing time-course studies, we investigated the dose-response
characteristics of neutrophilic alveolitis after IT LPS instillation.
Figure
2A
shows the percentage of alveolar macrophages and neutrophils in lung
lavage fluid 4 h after treatment with IT LPS (0, 0.001, 0.01, 0.1, 1.0, and 6.0 mg/kg). The threshold LPS dose for significant induction of
neutrophilic influx into the lung at 4 h was found to be 0.01 mg/kg. IT
administration of physiological pyrogen-free saline, the vehicle, was
not associated with the appearance of neutrophils in lung lavage fluid
at the 4-h time point. A dose of 0.001 mg/kg of LPS produced 11 ± 8% neutrophils in lavage fluid, but a dose of 0.01 mg/kg of LPS
produced 62 ± 4% neutrophils in lavage fluid at 4 h. IT LPS at
0.1, 1.0, and 6 mg/kg produced 61 ± 10, 71 ± 8, and 78 ± 3% neutrophils, respectively, at 4 h. Figure
2B shows the dose-response
relationship between IT LPS dose (0.001 and 0.01 mg/kg) and NF-B
binding activity in lung tissue nuclear protein extracts. In Fig.
2B, both bands A and C were
identified. Band A (intact Rel A/p50)
was faintly detected in lung samples from rats treated with 0.001 mg/kg
of IT LPS (Fig. 2B,
lanes
1-3) but was
not seen in samples from rats treated only with the normal saline
vehicle (data not shown). In contrast, both bands
A and C were
substantially upregulated in rat lungs after treatment with 0.01 mg/kg
of IT LPS (Fig. 2B, lanes
4-6). Similar
NF-
B binding activity was seen with larger doses of IT LPS (data not
shown). These data, together with our initial studies, show that
activation of NF-
B in lung tissue mirrors the influx of neutrophils
into lavagable air spaces after IT LPS instillation.
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To better identify which cells were responsible for the measured
NF-B activation in the lungs after IT LPS instillation, we measured
NF-
B binding activity in nuclear extracts of lung lavage fluid cells
after IT LPS instillation. We performed lung lavages with a total of 35 ml of sterile saline, combined the lavage fluid cells from two
different rats in each treatment group, and extracted nuclear proteins.
Figure
3A
shows the differential cell counts from an experiment in which 0.1 mg/kg of LPS was given intratracheally and the lungs were lavaged at
various time points. In this experiment, there were few (<10%)
neutrophils in the lavage fluid from 0 to 2 h after IT LPS
instillation, and 77% neutrophils were present at 4 h. In the lavage
fluid cells, NF-
B binding activity did not directly correlate with
neutrophilic influx (Fig. 3B). In
contrast to lung tissue NF-
B binding activity, lavage fluid cells
exhibited two peaks of activity. There was an initial increase in
NF-
B binding activity 15 min after IT LPS instillation (Fig.
3B, lane
2) that was diminished by 30 min and remained low until 4 h, when a second peak of NF-
B activity was found (Fig. 3B, lane
6). The initial peak of NF-
B binding activity
appears to be in alveolar macrophages because >90% of lavage fluid
cells were macrophages at that time point. No increase in NF-
B
binding activity was found in whole lung nuclear protein extracts 15 min after IT LPS instillation (data not shown). At 4 h, it is likely that the detectable NF-
B binding activity was from neutrophils because these cells accounted for 77% of lavagable cells at this time
point. Similar results were obtained when this experiment was repeated
with 6 mg/kg of IT LPS except that the second peak of lavage fluid cell
NF-
B binding activity and neutrophilic alveolitis were present 2 h
after 6 mg/kg of IT LPS (data not shown). Figure 3B also shows cold and nonspecific
competition (lanes 7 and
8, respectively), indicating specific
nuclear protein binding to the NF-
B motif because most of the band
is eliminated by adding excess unlabeled specific (cold) probe, but the
band is not eliminated by adding the same amount of nonspecific probe.
Although NF-
B bands
A-C are not well
separated in Fig. 3B,
lane 9 shows that the band contains
mostly Rel A p65 because antibodies to this NF-
B component result in
diminution of this band. In summary, these data show that IT LPS
results in early activation of NF-
B in alveolar macrophages that is
short-lived, followed by a subsequent peak of NF-
B activity in lung
tissue and in invading neutrophils that mirrors the alveolar
neutrophilic influx. It seems unlikely that the NF-
B binding
activity seen at later time points in entirely due to neutrophil
NF-
B activation, but neutrophil NF-
B activation may lead to a
positive feedback loop where NF-
B-dependent inflammatory mediator
production may lead to further neutrophil influx and activation.
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We used a multiprobe RPA and Northern blots to correlate NF-B
binding activity with cytokine gene expression in the lung after IT LPS
instillation. Figure 4 shows steady-state
mRNA levels for a variety of cytokines after treatment with IT LPS (6 mg/kg) with RPA. After treatment with IT LPS, expression of IL-1
mRNA, IL-1
mRNA, and TNF-
mRNA was apparent by 30 min and
persisted to 6 h. Subtle expression of IL-6 mRNA was evident by 1 h,
with more intense expression at 2, 4, and 6 h. TNF-
, IL-2 to -5 and -10, and interferon-
mRNAs were not detectable in lung tissue after
IT LPS treatment. L32 and glyceraldehyde-3-phosphate dehydrogenase are
products of constitutive genes that are illustrated to show equal
loading of RNA. In addition to this cytokine panel, we measured mRNA
levels of the rat C-X-C chemokine CINC, which Blackwell and colleagues
(1, 3, 5) and others (7, 9, 18) have shown to be an
important neutrophil chemoattractant in the rat.
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CINC mRNA expression as assessed by Northern blot is shown in Fig.
5. The kinetics of CINC gene expression in
lung tissue (Fig. 5A) were similar
to those of IL-6. CINC mRNA was upregulated 2 h after IT LPS
instillation and persisted until 6 h after treatment with IT LPS, with
minimal detectable CINC mRNA before 2 h. In dose-response studies (Fig.
5B), CINC mRNA was increased 4 h
after doses of IT LPS ranging from 0.01 to 1 mg/kg but was only
minimally detected in the lungs of one of two rats after treatment with 0.001 mg/kg of IT LPS.
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In these studies, expression of non-NF-B-dependent cytokines (IL-3
to -5 and -10 and interferon-
) was not induced in lung tissue by IT
LPS. Two potentially NF-
B-dependent cytokines, TNF-
and IL-2,
were not detectable. Other cytokines thought to be NF-
B dependent
showed two different patterns. First, CINC and IL-6 gene expression in
lung tissue correlated well with both lung tissue NF-
B binding
activity and neutrophilic alveolitis after IT injection of LPS. Second,
IL-1
, IL-1
, and TNF-
showed increased expression by 30 min
after IT LPS instillation, before NF-
B binding activity in lung
tissue but consistent with alveolar macrophage NF-
B activation,
suggesting alveolar macrophages as a major source of these cytokines.
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DISCUSSION |
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Our studies, like those of Ulich and colleagues (17-19,
21-24) and other investigators (16, 25, 26), show that IT
administration of LPS is associated with lung cytokine production and
neutrophilic influx. In addition, we have shown that IT LPS results in
an early and intense neutrophilic influx that is closely associated
with NF-B binding activity in lung tissue. Both time-course and
dose-response studies showed a close relationship between lung NF-
B
binding activity, CINC gene expression, and neutrophilic alveolitis. We also discovered that the gene expression of several NF-
B-dependent cytokines in lung tissue was increased after IT LPS instillation and
that the production of early proinflammatory cytokines, TNF-
, IL-1
, and IL-1
, preceded peak NF-
B binding activity in lung tissue. Increased mRNA of these cytokines correlated much better with
the early NF-
B binding activity that was identified in alveolar macrophages. Because alveolar macrophages are known to produce all
three of these cytokines (15), it is likely that macrophages are a
major source of these cytokines after IT instillation of LPS. This
early production of cytokines (IL-1 and TNF-
) may be an early
response by alveolar macrophages that initiates the cytokine cascade
and signals other cells in the lung to produce "distal" cytokines
such as CINC and other chemokines that are important for signaling
neutrophilic immigration. Lung epithelial cells, for example, are known
to produce IL-8 in culture at much higher concentrations in response to
TNF-
and IL-1 than in response to LPS (14).
Another interesting finding of the present study is the late appearance
of NF-B DNA binding activity in lavage fluid cells, corresponding to
the time of neutrophil predominance in the lavage fluid cell
population. Although neutrophils previously have been shown to activate
NF-
B and produce inflammatory mediators (10), our results suggest
that neutrophils in this model use NF-
B-dependent mechanisms to
produce inflammatory mediators and amplify the inflammatory response.
In combination, these data suggest that a complex pattern of cellular
NF-
B activation and cytokine production coordinates neutrophilic
alveolitis after IT LPS instillation.
Further insight into the systemic and local mechanisms of acute
pulmonary inflammation via direct and indirect inflammatory stimuli may
help to clarify the sequence of events leading to neutrophilic lung
inflammation and lung injury. Key control points in the inflammatory
cascade, such as NF-B, may provide targets for intervention to
modulate inflammation by local action in the lungs, targeting specific
cell types or, systemically, targeting the balance between lung and
systemic inflammatory mediator production. Our data suggest that agents
that prevent activation of NF-
B in alveolar macrophages, if given
early, could prevent propagation of the inflammatory cascade. In
contrast, agents given later that have a broader cellular target in the
lung could decrease the intensity of neutrophilic inflammation by
preventing more distal elements of the inflammatory cascade.
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ACKNOWLEDGEMENTS |
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This work was supported by the Department of Veterans Affairs; a Parker B. Francis Foundation Fellowship in Pulmonary Research; The American Lung Association, and National Heart, Lung, and Blood Institute Grant HL-07123.
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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: T. S. Blackwell, Division of Allergy, Pulmonary, and Critical Care Medicine, Vanderbilt Univ. School of Medicine, T-1217 MCN, Nashville, TN 27232-2650 (E-mail: timothy.blackwell{at}mcmail.vanderbilt.edu).
Received 20 January 1999; accepted in final form 26 May 1999.
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