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B Activity in the Bronchi of an Animal Model of Asthma1
Departments of
*
Physiology and
Immunology/Vaccinology, Faculty of Veterinary Medicine, and
Laboratory of Medical Chemistry/Medical Oncology, Faculty of Medicine, University of Liege, Liege, Belgium
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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B induces many
inflammatory proteins as well as its own inhibitor, IB-
, thus
assuring a transient response upon stimulation. However,
NF-B-dependent inflammatory gene expression is persistent in
asthmatic bronchi, even after allergen eviction. In the present report
we used bronchial brushing samples (BBSs) from heaves-affected horses
(a spontaneous model of asthma) to elucidate the mechanisms by which
NF-B activity is maintained in asthmatic airways. NF-B activity
was high in granulocytic and nongranulocytic BBS cells. However,
NF-B activity highly correlated to granulocyte percentage and was
only abrogated after granulocytic death in cultured BBSs. Before
granulocytic death, NF-B activity was suppressed by simultaneous
addition of neutralizing anti-IL-1
and anti-TNF- Abs to
the medium of cultured BBSs. Surprisingly, IB-, whose expression
is not regulated by NF-B, unlike IB-, was the most prominent
NF-B inhibitor found in BBSs. The amounts of IB- were low in
BBSs obtained from diseased horses, but drastically increased after
addition of the neutralizing anti-IL-1 and anti-TNF- Abs.
These results indicate that sustained NF-B activation in asthmatic
bronchi is driven by granulocytes and is mediated by IL-1 and
TNF-. Moreover, an imbalance between high levels of IL-1- and
TNF--mediated IB- degradation and low levels of IB-
synthesis is likely to be the mechanism preventing NF-B deactivation
in asthmatic airways before granulocytic death. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B, AP-1, NF-AT, cAMP response element
binding protein, STATs, and GATA-3 (Refs 2 and
3 ; for review, see Ref. 4). All of the
inflammatory genes overexpressed in asthma, such as those encoding
proinflammatory cytokines, chemokines, adhesion molecules, and
inflammatory enzymes, contain B sites for NF-B within their
promoter (for review, see Ref. 5), suggesting that these
genes are controlled predominantly by NF-B and that NF-B could be
of particular importance in the initiation and the perpetuation of
allergic inflammation. This assumption is reinforced by the fact that
glucocorticosteroids, the most potent treatment for asthma, strongly
inhibit NF-B in vitro (6, 7).
The NF-B family is composed of five structurally related DNA-binding
proteins, called p50, p52, p65/RelA, c-Rel/Rel, and RelB (for review,
see Ref. 8). The most common form of NF-B is a
heterodimer composed of p50 and p65 subunits, although the different
family members can associate in various homo- or heterodimers through a
highly conserved N-terminal sequence, called the Rel homology domain.
Dimerization of various NF-B subunits produces complexes with
different DNA-binding specificities and trans activation
potentials. In most cell types, inactive NF-B complexes are
associated with inhibitory proteins of the IB family, which
sequester NF-B in the cytoplasm. The members of the IB family are
IB-, IB-, IB-
, p100, p105, and Bcl-3, where the most
common IB protein is IB- (8, 9). p105 and p100
are the precursors of p50 and p52, respectively. Following various
stimuli, such as viruses, bacteria, pro-oxidants, and proinflammatory
cytokines, IB proteins are first phosphorylated, ubiquitinated, and
then rapidly degraded by the proteasome, allowing NF-B nuclear
translocation and transcriptional initiation of NF-B-dependent genes
(10).
Macrophages of induced-sputum and bronchial epithelial cells from
stable asthmatic patients exhibit increased NF-B activity compared
with cells from healthy patients (11). Mice deficient in
p50 or c-Rel are unable to develop eosinophilic airway inflammation
when sensitized and challenged with OVA (12, 13). In
bronchial brushing samples
(BBSs)3 recovered in
heaves-affected horses, a spontaneous animal model of asthma (for
review, see Ref. 14), NF-B complexes are mainly
atypical p65 homodimers (15). p65 homodimer activity
drastically increases in BBSs from heaves-affected horses challenged
with moldy hay, which contains the allergens responsible for the
disease (i.e., proteins borne by spores of Asperigillus
fumigatus, Faenia rectivirgula, and Thermoactinomyces
vulgaris). Interestingly, this increased activity is maintained at
high or moderate levels for at least 21 days after allergen eviction
from the horses’ environment. In this model it has also been
demonstrated that p65 homodimer activity found in BBSs is highly
correlated to the degree of lung dysfunction and to the level of ICAM-1
expression (15). Although these in vivo observations
confirmed that NF-B is likely to play a crucial role in allergic
inflammation and in subsequent airway obstruction, no study was devoted
to the mechanisms by which NF-B activity is maintained in lung cells
even when the etiologic agent is absent.
In the present report, we describe studies aimed at identifying the
mechanisms by which NF-B activity is regulated in cells obtained by
bronchial brushing in heaves-affected horses after allergen eviction,
and we propose a cellular and molecular model that accounts for the
persistent NF-B activity observed in the bronchi of this animal
model of asthma.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Nine horses (564 ± 83 kg; 18.6 ± 1.8 years; mean ± SD) with a history and clinical signs of heaves were used. These horses typically developed acute airway obstruction (crisis) when housed in a barn and fed moldy hay, and they entered clinical remission once pastured or stabled in a controlled environment. One month before the experiment they underwent a thorough clinical examination, including an electrocardiogram, arterial blood gas analysis, hematology, endoscopy of the airways, tracheo-bronchial lavage, and pulmonary scintigraphy. This confirmed that they suffered from heaves and were free from any other health problems. Six healthy horses (605 ± 99 kg; 7.6 ± 2.6 years) were used as controls. Experimental horses did not receive any treatment during the month preceding the experiments.
Bronchial cells of heaves-affected horses were obtained by bronchial
brushing on two separate occasions: 24 h after the onset of a
crisis and then 21 days after removal from the causative environment.
To obtain crisis, the horses were stabled and subjected to a natural
challenge with moldy hay. The horses were considered to be in crisis
when their breathing mechanic variables were within the following
limits: maximal difference in pleural pressure 
2.00 kPa, total
pulmonary resistance 0.2 kPa/l · s-1, and
dynamic compliance 
8 l/kPa-1. These
respiratory mechanic variables were calculated from simultaneous
measurements of esophageal pressure, air flow, and tidal volume (for
more technical details, see Ref. 16). Eviction of the
antigenic agents was obtained by pasturing the horses or stabling them
with dust-free bedding and feed. Healthy horses were investigated twice
at a 21-day interval. The protocol was approved by the ethics committee
of the University of Liege.
Bronchial brushings
Horses were premedicated i.v. with 0.01 mg/kg romifidine (Sedivet; Boehringer Ingelheim, Ingelheim, Germany). Bronchoscopy was performed with a 9-mm diameter bronchoscope (Pentax, Breda, The Netherlands) using a transnasal approach. The brushing was performed in 10 different places, from the main bronchi to the fourth generation airways, by inserting a cytology brush (Cook Veterinary Products, Eight Mile Plains, Australia) into the different segments. Bronchial cells were obtained using 20 gentle upward and downward strokes of the brush against the airway walls. Care was taken to avoid bleeding. Bronchi were not irrigated with physiological serum before brushing, to conserve leukocytes and to ensure that samples were representative of the cellular changes occurring within the bronchi of diseased horses.
Cell processing
After retraction of the brush into its protective sheath and its removal from the bronchoscope channel, collected cells were dislodged by shaking the brush into 15-ml conical tubes containing ice-cold RPMI 1640 medium (Life Technologies, Merelbeke, Belgium) supplemented with 1% glutamine, 10% FBS, 50 µg/ml gentamicin, and 10 µg/ml amphotericin B. The harvested cell suspension was vortexed and filtered through gauze to remove mucus. The cells were then centrifuged at 800 x g for 5 min, and the pellet was resuspended in LHC-8 complete medium without hydrocortisone (Biofluids, Rockville, MD) supplemented with 10 µg/ml amphotericin B. The cells were then incubated at 37°C in a 5% CO2-95% air mixture for different times before protein extraction. The minimal culture time before protein extraction was 3 h. Cell density was assessed by the use of a hemocytometer, and cell viability was evaluated by propidium iodide exclusion (5 µg/ml of culture medium). Cell differentials were performed on cytospin preparations stained with Diff-Quick (Dade Behring, Dudingen, Germany). Where necessary, polymorphonuclear cells were separated from the other cells using Histopaque centrifugation (specific gravity, 1.077; Sigma, Bornem, Belgium).
Cytoplasmic and nuclear protein extraction
Cytoplasmic and nuclear protein extracts were prepared as previously described (17). Cytoplasmic buffer contained 10 mM HEPES (pH 7.9), 10 mM KCl, 2 mM MgCl2, 0.1 mM EDTA, 0.2% (v/v) Nonidet P-40, and 1.6 mg/ml protease inhibitors (Complete; Roche, Mannheim, Germany). The pelleted nuclei were resuspended in 20 mM HEPES (pH 7.9), 1.5 mM MgCl2, 0.2 mM EDTA, 0.63 M NaCl, 25% (v/v) glycerol, and 1.6 mg/ml protease inhibitors (nuclear buffer), incubated for 20 min at 4°C, and centrifuged for 30 min at 12,000 x g (Eppendorf centrifuge 5415C; Eppendorf Scientific, Hamburg, Germany). Protein concentrations were quantified with the Micro bicinchoninic acid protein assay reagent kit (Pierce, Rockford, IL).
Anti-IB Abs
The anti-IB Abs used were 1) a mouse mAb directed against
IB- (a gift from Katrina Wood, University of Oxford, Oxford,
U.K.); 2) a rabbit polyclonal Ab recognizing an
NH2-terminal peptide of mouse IB- (Santa
Cruz Biotechnology, Santa Cruz, CA); 3) a mouse mAb directed against aa
1-444 of the human p52 subunit (Upstate Biotechnology, Lake Placid,
NY); and 4) a rabbit polyclonal Ab recognizing an
NH2-terminal peptide (aa 1–12) of human p50
(Upstate Biotechnology). Immunoblot experiments performed with
cytoplasmic extracts prepared from equine lymphocytes showed that all
these Abs are equine reactive.
Plasmids
The pRc/CMV-hemagglutinin (HA)-IB- expression vector was
provided by Alain Israël and Robert Weil (Institut Pasteur,
Paris, France). The empty pRc/CMV plasmid was purchased from Invitrogen
(San Diego, CA). Constructions were linearized with ScaI
before coupled in vitro transcription and translation.
Coupled in vitro transcription and translation of IB-
Linearized pRc/CMV and pRc/CMV-HA-IB- plasmids were in
vitro transcribed and translated simultaneously using the TnT T7
Coupled Wheat Germ Extract System (Promega, Madison, WI) according to
the manufacturer’s instructions. The reactions were performed either
with or without [35S]methionine in the
transcription-translation mixture. The
[35S]methionine-labeled translated products
were analyzed by electrophoresis in a 10% polyacrylamide-SDS gel and
autoradiography to verify that they contained proteins of the expected
molecular mass of 
47 kDa for HA-IB- (see Ref. 18
for the first characterization of IB-). Unlabeled translated
products were used in EMSA experiments.
EMSAs
Binding reactions were performed for 30 min at room temperature
with 5 µg of nuclear proteins in 20 mM HEPES (pH 7.9), 10 mM KCl, 0.2
mM EDTA, 20% (v/v) glycerol, 1% (w/v) acetylated BSA, 3 µg of
poly(dI-dC) (Amersham Pharmacia Biotech, Aylesbury, U.K.), 1 mM DTT, 1
mM PMSF, and 100,000 cpm of 32P-labeled
double-stranded oligonucleotide probes. Probes were prepared by
annealing the appropriate single-stranded oligonucleotides
(Eurogentech, Liege, Belgium) at 65°C for 10 min in 10 mM Tris, 1 mM
EDTA, and 10 mM NaCl, followed by slow cooling to room temperature. The
probes were then labeled by end-filling with the Klenow fragment of
Escherichia coli DNA polymerase I (Roche), with
[32P]dATP and [32P]dCTP
(DuPont-New England Nuclear, Les Ulis, France). Labeled probes were
purified by spin chromatography on Sephadex G-25 columns (Roche).
DNA-protein complexes were separated from unbound probe on 4% native
polyacrylamide gels at 150 V in 0.25 M Tris, 0.25 M sodium borate, and
0.5 mM EDTA, pH 8.0. Gels were vacuum dried and exposed to Fuji x-ray
film (Tokyo, Japan) at -80°C for 12 h. The amount of specific
complexes was determined by photodensitometry of the autoradiography
(Gel Doc 2000; Bio-Rad, Hercules, CA). To confirm specificity,
competition assays were performed with a 50-fold excess of unlabeled
wild-type and mutated probes. The sequences of the oligonucleotides
used in this work were as follows: wild-type palindromic B probe
(19), 5'-TTGGCAACGGCAGGGGAATTCCCCTCTCCTTAGGTT-3'; and
mutated palindromic B probe,
5'-TTGGCAACGGCAGATCTATTCCCCTCTCCTTAGGTT-3'.
For experiments performed with in vitro translation products, 2 µl of each reaction was incubated with the nuclear extracts obtained from BBSs for 30 min either before or after incubation with the radiolabeled probe.
Immunoblots
Protein extracts (10 µg) were added to a loading buffer (10 mM
Tris-HCl (pH 6.8), 1% (w/v) SDS, 25% (v/v) glycerol, 0.1 mM 2-ME, and
0.03% (w/v) bromophenol blue), boiled, and run on a 10% SDS-PAGE gel.
After electrotransfer to polyvinylidene difluoride membranes (Roche)
and blocking overnight at 4°C with 20 mM Tris (pH 7.5), 500 mM NaCl,
0.2 (v/v) Tween 20 (Tris-HCl/Tween), and 5% (w/v) dry milk, the
membranes were incubated for 1 h with the first Ab (1/250 dilution
for IB- and 1/1000 dilution for the other IB proteins),
washed, and then incubated for 45 min with peroxidase-conjugated rabbit
anti-mouse IgG (1/2000 dilution) for IB- and p52 (Dako,
Glostrup, Denmark), or peroxidase-conjugated goat anti-rabbit IgG
(1/5000 dilution) for the other IB proteins (Kirkegaard & Perry,
Gaithersburg, MD). The results of the reaction were revealed with the
enhanced chemiluminescence detection method (ECL kit; Amersham
Pharmacia Biotech). Equal loading of protein on the gels was confirmed
in all experiments by probing the blots for either -tubulin (Santa
Cruz Biotechnology) in the case of cytoplasmic extracts or Oct-1 (Santa
Cruz Biotechnology) in the case of nuclear extracts (data not
shown).
Neutralization experiments
Neutralizing Abs directed against recombinant human IL-1 and
TNF- were purchased from Sigma. Anti-IL-1 and anti-TNF-
Abs were used at 3 or 8 µg/ml. These were incubated for 180 min
before protein extraction with 3-h cultured BBSs obtained from
heaves-affected horses, 21 days after allergen eviction.
Statistical analysis
Linear associations between variables were assessed by the use of standard least-square linear regressions. Correlation coefficients (r) were presented as measures of linear association for regression relationships. Significant differences of the slopes from zero were determined using two-tailed Student’s t test. The differences between mean values were estimated using Student’s t tests for unpaired data. p < 0.05 was considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The number of harvested cells averaged 19.9 ± 7.0 (mean
± SD) million cells/animal (range, 11–34 million). Differential cell
counts showed a significant increase in the percentage of granulocytes
in BBSs obtained from heaves-affected horses compared with healthy
horses (Table I
). The viability of
harvested granulocytes (92.5 ± 4.2%), as determined by propidium
iodide exclusion, was significantly greater than the viability of the
other cells present in BBSs (24.2 ± 9.3%). Accordingly, total
cell viability measured in BBSs from heaves-affected horses was
significantly higher than that measured in BBSs from healthy horses
(Table I). We previously reported lower percentages of granulocytes in
BBSs from heaves-affected horses (i.e., 3.4 ± 0.7% during the
crisis and 1.8 ± 1.3% 21 days after allergen eviction), and cell
viability that was not significantly different between healthy and
diseased horses (15). This could be imputed to the fact
that bronchi were irrigated with physiological serum before brushing in
this earlier study. Indeed, bronchial irrigation partly eliminates
granulocytes, which are present in large quantities in diseased horses
at the surface of the airway epithelium.
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B activity in BBSs
Consistent with our previous studies (15), NF-B
activity was much greater in nuclear extracts prepared from BBSs of
heaves-affected horses during crisis (Fig. 1A, lanes 4,
6, and 8), when compared with extracts from BBSs
of healthy horses (Fig. 1A, lanes 1–3).
Twenty-one days after the eviction of the causative agents, NF-B
activity was maintained at high or moderate levels in BBSs from
diseased horses (Fig. 1A, lanes 5, 7,
and 9). As the percentage of granulocytes and NF-B
activity simultaneously increased in BBSs from heaves-affected horses,
notably during crisis, correlations between the percentage of
granulocytes in BBSs and the intensity of NF-B DNA binding, as
measured by photodensitometry, were calculated. These regression
analyzes were conducted with the results obtained from four separate
EMSAs. Correlation coefficients between the percentage of granulocytes
and the intensity of specific NF-B bands were 0.96
(p < 0.001, first gel), 0.97
(p < 0.001, second gel), 0.93
(p < 0.01, third gel), and 0.89
(p < 0.01, fourth gel). These significant
correlations were all positive. Results obtained from a representative
gel are shown in Fig. 1.
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B activity is increased in both granulocytes and
nongranulocytic cells contained in BBSs from heaves-affected horses
The strong correlation between the intensity of NF-B activity
and the percentage of granulocytes presents in BBSs suggested that the
increased NF-B activity observed in BBSs from heaves-affected horses
was restricted to polymorphonuclear cells. To verify this hypothesis,
BBSs were recovered in heaves-affected horses 21 days after allergen
eviction, and the granulocytes were separated from the other BBS cells
using Histopaque centrifugation. Because BBSs contained many clusters
made of various cell types, the use of specific immunological methods,
such as flow cytometry, to separate the granulocytes from the other BBS
cells was inadequate. Histopaque centrifugation only allowed partial
cell separation. Two fractions were obtained: a granulocyte-enriched
fraction, in which the percentage of granulocytes averaged 62.1 ±
12.2%, and a bronchial epithelial cell (BEC)-enriched fraction, in
which the percentage of granulocytes averaged 9.5 ± 9.9%. EMSAs
performed with total and enriched samples obtained simultaneously from
the same heaves-affected horses showed identical NF-B activities
(Fig. 2), indicating that NF-B
activity is similarly increased in both granulocytes and
nongranulocytic cells contained in BBSs from diseased horses.
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B deactivation are concomitant in
cultured BBSs from heaves-affected horses
NF-B activity was maintained at high or moderate levels in the
bronchi of heaves-affected horses 21 days after allergen eviction,
indicating that NF-B activity in asthma-like diseases does not
necessarily require the continuous presence of the etiologic agent. To
determine whether increased NF-B activity is also sustained ex vivo,
three BBSs obtained simultaneously from the same heaves-affected horses
(n = 9) 21 days after the crisis were cultured for 3,
24, or 48 h before assessment of NF-B-binding activity. Nuclear
extracts prepared from BBSs cultured for 3 and 24 h demonstrated
identical NF-B activities (examples are given in Fig. 3A, lanes 1,
2, 4, and 5). BBSs cultured for
48 h displayed NF-B activities that were either similar to
those observed at 3 and 24 h (n = 5; an example is
provided in Fig. 3A, lane 6) or drastically
decreased (n = 4; an example is given in Fig. 3A, lane 3).
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B, lane 6), whereas
granulocytic viability considerably decreased in BBSs from the four
other horses (an example is provided in Fig. 3B, lane
3). Reduced granulocytic viability was always accompanied by a
drastic decrease in NF-B activity (an example is given in Fig. 3, A and B, lane 3). Conversely, when
granulocytic viability was maintained at high levels, NF-B activity
was also sustained at high levels (an example is given in Fig. 3, A and B, lane 6). The link between
granulocytic death and abolishment of NF-B activity in cultured BBSs
from heaves-affected horses was confirmed by regression analyses, which
were conducted with the results obtained from three separate EMSAs.
Correlation coefficients between granulocyte viability and the
intensity of NF-B bands were 0.93 (p <
0.01, first gel), 0.89 (p < 0.01, second gel),
and 0.87 (p < 0.05, third gel). Correlation
coefficients between the viability of the other BBS cells and NF-B
activity were also calculated. These correlation coefficients were not
significant and ranged from 0.29–0.39. Results obtained from a
representative gel are shown in Fig. 3. These data show that sustained
NF-B activity in cultured BBSs requires the presence of living
granulocytes.
IB- is the most prominent IB protein found in equine BBSs
In most cells stimulation leading to IB- proteolysis and
nuclear translocation of NF-B also results in the subsequent rapid
NF-B-dependent induction of IB- (20, 21). The
reaccumulation of IB- following its loss allows a fast repression
of NF-B activity, thereby ensuring a transient NF-B response. In
cultured BBSs from heaves-affected horses, NF-B activity was
maintained as long as living granulocytes were present (Fig. 3),
suggesting that mutual regulation of NF-B and IB- is impaired
in these samples. Two hypotheses could account for this observation:
either IB- is degraded as soon as it is resynthesized, preventing
NF-B deactivation, or IB- is not produced in the BBSs obtained
from heaves-affected horses. To explore these hypotheses, the presence
of all IB proteins in cytoplasmic and nuclear extracts prepared from
3-h cultured BBSs of healthy and heaves-affected horses 21 days after
allergen eviction was assessed by immunoblots.
Only very low amounts of IB- were detected in cytoplasmic and
nuclear extracts obtained from BBSs of both healthy and heaves-affected
horses (Fig. 4). Similarly, p100 was
undetectable (data not shown). Only small amounts of p105, the
precursor of p50, were revealed by immunoblot in cytoplasmic extracts
obtained from cells of healthy and heaves-affected horses (data not
shown). On the contrary, significant amounts of IB- were observed
in cytoplasmic extracts from BBSs of healthy horses, while considerably
lower amounts of cytoplasmic IB- were observed in BBSs obtained
from heaves-affected horses (Fig. 4). IB- was not detectable in
the nuclear extracts from BBSs of healthy and heaves-affected horses
(Fig. 4). These results indicate that IB-, rather than IB-,
is the most prominent IB protein found in the BBSs of horses and
suggest that IB- is degraded in BBSs from heaves-affected
horses.
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and anti-TNF- Abs inhibit the
persistent IB- degradation and NF-B activation in BBSs from
heaves-affected horses
NF-B stimulates the production of IL-1 and TNF-. These
proinflammatory cytokines induce the degradation of the IB proteins
and the subsequent activation of NF-B, thus initiating
autoregulatory feedback loops (for review, see Ref. 22).
To determine whether these loops were involved in the sustained
IB- degradation and NF-B activation observed in the BBSs of
heaves-affected horses before granulocytic death, neutralizing
anti-IL-1 and/or anti-TNF- Abs were added to the medium
of 3-h cultured BBSs obtained from heaves-affected horses 21 days after
allergen eviction. The final Ab concentrations was either 3 or 8
µg/ml. Cytoplasmic and nuclear extracts were prepared from treated
BBSs 180 min after Ab addition and were subsequently analyzed for IB
protein expression by immunoblot and for NF-B binding activity by
EMSA. Neither the addition of anti-IL-1 Abs (3 or 8 µg/ml) nor
the addition of anti-TNF- Abs (3 or 8 µg/ml) was able to
reduce IB- degradation and NF-B activation in BBSs (Fig. 5A). Conversely, when added
simultaneously, the anti-IL-1 and anti-TNF- Abs (3
µg/ml of each Ab) drastically increased the cytoplasmic and nuclear
amounts of IB- (Fig. 5B) and markedly decreased the
NF-B activity in BBSs (Fig. 5A), indicating that
autoregulatory feedback loops involving both IL-1 and TNF- are at
least partly responsible for the sustained IB- degradation and
NF-B activation in the cultured BBSs from diseased horses. The
cytoplasmic and nuclear amounts of IB-, p100, and p105 were not
altered by the addition of neutralizing Abs to the culture medium.
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B- prevents p65 homodimer DNA binding and removes bound p65
homodimers from B sites
We had previously demonstrated that active NF-B complexes found
in BBSs of heaves-affected horses were mainly p65 homodimers, rather
than classical p65-p50 heterodimers (15). Knowing that
IB- is the most prominent IB protein present in equine BBSs
(Fig. 4) and that the appearance of IB- in the nucleus of BBS
cells and NF-B deactivation are concomitant (Fig. 5), we
hypothesized that IB- is able to prevent p65 homodimer DNA
binding and to displace bound p65 homodimers from their B sites. To
verify this hypothesis, the effects of recombinant IB- on p65
homodimer DNA binding were investigated using EMSAs. First, either the
linearized plasmid vector pRc/CMV or this same linearized plasmid
containing the IB- cDNA insert (pRc/CMV-HA-IB-) was used as
DNA templates in a coupled in vitro transcription/translation reaction
system. The [35S]methionine-labeled translated
products were analyzed by electrophoresis in a 10% polyacrylamide-SDS
gel and by autoradiography. A major translation product of 47 kDa
was synthesized from pRc/CMV-HA-IB-, consistent with the
predicted size of HA-IB- (Fig. 6A). Two microliters of each
unlabeled lysate was then incubated for 30 min with nuclear extracts
obtained from BBSs of heaves-affected horses, either before or after
incubation with the radiolabeled B probe. Afterward, DNA-protein
complexes were analyzed by EMSA. Mock-translated products were unable
to alter p65 homodimer DNA-binding (Fig. 6B, lanes
2 and 3). On the contrary, in vitro translated
IB- was able to prevent p65 homodimer DNA binding and remove
bound p65 homodimers from their B sites (Fig. 6B,
lanes 4 and 5, respectively).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B activity, which
plays a key role in the transcriptional initiation of many inflammatory
genes, drastically increases in the bronchi of heaves-affected horses
after allergen exposure and is maintained at high or moderate levels in
the bronchi of diseased horses for at least 21 days after allergen
eviction from their environment (15). Here, we show that
this increased NF-B activity also persists for at least 24–48 h in
cultured BBSs obtained from heaves-affected horses 21 days after
allergen eviction. As NF-B activity usually returns to basal level
within a few hours after the removal of external stimuli
(23), our observations indicate that NF-B activity is
aberrantly sustained in the bronchial cells of heaves-affected horses
after allergen eviction and strongly suggest that this persistent
transcriptional activity could account for the maintained expression of
inflammatory genes in asthmatic bronchi.
The accumulation of active granulocytes in the airways is thought to be
of particular importance in the development of clinical asthma
(24). Indeed, several investigators have demonstrated a
positive and significant correlation between eosinophil profusion in
the airways and lung dysfunction in asthmatic patients (25, 26). Furthermore, the resolution of eosinophilic inflammation,
which depends upon eosinophil apoptosis, is associated with clinical
improvement of asthma (27). In the present study, the
levels of NF-B activity were high or moderate in granulocytes and
nongranulocytic cells contained in BBSs obtained from heaves-affected
horses 21 days after allergen removal. However, NF-B activity
strongly correlated with the percentage of granulocytes present in BBSs
and was completely abrogated after granulocytic death, suggesting that
the sustained NF-B activation that occurs in the airways of
heaves-affected horses is mainly driven by the inflammatory cells that
remain or appear in the bronchi after allergen eviction. As the level
of NF-B activity in the bronchi is closely related to the degree of
pulmonary dysfunction (15), our results also provide a new
insight into the molecular mechanisms by which the granulocytes impair
lung function. First, the level of NF-B activity in the granulocytes
probably determines the amounts of broncho- and vasoactive inflammatory
mediators released by these cells. Second, one may assume that
activated granulocytes also secrete cytokines that are able to initiate
the NF-B-dependent synthesis of inflammatory mediators by other
bronchial cells. Granulocytic death and clearance would be
prerequisites for the cessation of the direct and indirect effects of
these cells on lung function.
The physiological half-life of the circulating neutrophil, the most
abundant granulocyte, is only 6 h (for review, see Ref.
28). In the present study, granulocyte viability was much
longer, indicating that protective mechanisms delay inflammatory cell
death in asthmatic airways. TNF-, which was produced by BBSs from
heaves-affected horses, induces apoptosis of ex vivo cultured
neutrophils at early time points, but inhibits apoptosis after culture
for 18 h (29). This delayed protective effect is lost
when protein synthesis is inhibited, indicating that TNF- induces
anti-apoptotic proteins that protect neutrophils which avoid early
death (for review, see Ref. 30). It is likely that
inflammatory cells that invade the site of inflammation are those that
express anti-apoptotic proteins and are subsequently protected from
death. This possibility might explain the prolonged survival observed
in granulocytes from heaves-affected horses, even in the presence of
TNF-. A second hypothesis could account for the increased survival
of inflammatory cells from asthmatic bronchi. Indeed, many cytokines
present at inflammatory sites, such as GM-CSF, are able to delay
granulocyte apoptosis (for review, see Ref. 30). These
anti-apoptotic cytokines could counteract the cytotoxic effects of
TNF-.
An intriguing question concerns the maintenance of NF-B activity in
the bronchi before granulocytic death. A hallmark of many
NF-B-dependent genes that are switched on in inflammatory diseases
is that their expression can be induced by the proinflammatory
cytokines IL-1 and TNF- (31). Activated granulocytes
generate high amounts of IL-1 and TNF- (32, 33, 34).
IL-1 and TNF- activate NF-B, which, in turn, induces the
expression of these proinflammatory cytokines, thus initiating
autoregulatory feedback loops (for review, see Ref. 22).
Accordingly, we postulated that these autoregulatory feedback loops
might be involved in the granulocyte-mediated persistent NF-B
activity in bronchial cells of heaves-affected horses after allergen
eviction. Addition of both neutralizing anti-IL-1 and
anti-TNF- Abs to cultured BBSs from heaves-affected horses
resulted in the suppression of NF-B activity, confirming our
hypothesis. These findings are consistent with previous data from
Lentsch et al. (35), who showed that NF-B activity
occurs in a time course similar to that for the production of IL-1
and TNF- during IgG immune complex-induced lung injury.
Interestingly, when either the anti-IL-1 or anti-TNF- Abs
was added individually to the medium of cultured BBSs, each was
incapable of reducing NF-B activity. These results indicate that
either cytokine is independently able to maximally stimulate the
signaling pathway leading to NF-B activation in the cultured BBSs
from heaves-affected horses and emphasize that the antagonization of a
single cytokine would probably have a minor effect on allergic
inflammation.
IB-, rather than IB-, was the most prominent IB protein
found in BBSs from healthy and heaves-affected horses. Moreover, the
appearance of IB- in the nucleus of BBS cells from
heaves-affected horses was accompanied by NF-B deactivation, as
observed after treatment with anti-IL-1 and anti-TNF-
Abs. Finally, recombinant IB- was able to prevent DNA binding by
p65 homodimers, which are the most abundant NF-B complexes found in
equine BBSs (15) and was able to remove bound p65
homodimers from their B sites. Because BBSs contained mainly BECs in
healthy horses and BECs plus granulocytes in heaves-affected horses,
our results unambiguously demonstrate that the inhibition of p65
homodimers by IB- regulates NF-B-dependent gene expression in
equine bronchial epithelial cells and in equine bronchial granulocytes.
However, large amounts of IB- are observed in equine blood
granulocytes (our unpublished observations), suggesting a shift in
IB protein expression during granulocyte migration and activation.
Although unexpected, our results are in accordance with those of
Lentsch et al. (35, 36), who demonstrated that
deactivation of NF-B complexes predominantly composed of p65 by
secretory leukocyte protease inhibitor is associated with increased
levels of IB-, but not IB-, in a rat model of IgG immune
complex-induced alveolitis. Furthermore, previous in vitro studies have
demonstrated that IB- (37) as well as IB-
(38) preferentially inhibit the p65 homodimeric form of
NF-B. As no equine reactive anti-IB- Ab is available, the
inhibitory function of IB- in BBSs from heaves-affected horses
was not investigated.
IB- has been demonstrated to be involved in the persistent
NF-B activity observed in some cells, including activated 70Z/3
pre-B cells (39), WEHI 231 mature B cells
(40), HIV-1-infected myeloid cells (41), and
T cells infected by the human T cell leukemia virus type 1
(42). In human T cell leukemia virus type 1-infected T
cells, the persistent NF-B activity results from the continuous
degradation of IB- by the virally encoded Tax protein
(42). In the other cells stimulation results in the
degradation of IB-, which is resynthesized in a
hypophosphorylated form that sustains NF-B activation
(39, 40, 41). Indeed, hypophosphorylated IB- interacts
with NF-B without masking its nuclear localization signal and its
DNA binding site, thus acting as a chaperone for NF-B nuclear entry
and activity. Furthermore, hypophosphorylated IB- prevents
NF-B resequestration by IB-. However, IB- was not
associated with active p65 homodimers in the nucleus of BBS cells
obtained from heaves-affected horses, indicating that
hypophosphorylated IB- is not involved in the sustained NF-B
activity found in these cells.
Rapid NF-B-dependent resynthesis of IB- establishes an
autoregulatory loop by which NF-B activation is self-limited (Refs.
20 and 21 ; for review, see Ref.
43). Conversely, NF-B activation does not induce
IB- overexpression, indicating that NF-B complexes exclusively
released from IB- are not inhibited by an autoregulatory feedback
mechanism (18). This observation led Thompson et al.
(18) to anticipate that activation of NF-B would
probably be persistent in tissues lacking IB-, because no
feedback inhibition through increased synthesis of IB- would
occur in these tissues. In the present report, IB- was lacking,
and IB- was continuously degraded by proinflammatory cytokines in
BBSs from heaves-affected horses, providing the first in vivo example
of the mechanism of sustained NF-B activity theoretically described
by Thompson et al. (18).
Taken together, our results allow us to propose a cellular and
molecular model that accounts for the persistent NF-B activity
occurring in the bronchi of heaves-affected horses after allergen
eviction (Fig. 7). In this model the
NF-B activity is maintained as long as there are living granulocytes
in the airways. Before granulocytic death, NF-B activity is
maintained in all bronchial cells by granulocyte-dependent
autoregulatory feedback loops involving the proinflammatory cytokines
IL-1 and TNF-. IB- is expressed at a basal level unaffected
by cell stimulation with IL-1 and TNF-, is rapidly degraded, does
not reach the nucleus, and is subsequently unable to stop the
cytokine-mediated NF-B activation. After granulocytic death, the
autoregulatory feedback loops involving IL-1 and TNF- are
strongly attenuated, allowing IB--dependent NF-B deactivation
in nongranulocytic cells.
|
|
Acknowledgments |
|---|
B-; Drs. Alain Israël and
Robert Weil (Institut Pasteur, Paris, France) for the
pRc/CMV-HA-IB- expression vector; Drs. Pierre Chatelain, Jacques
Gielen, Renaud Louis, Roy Massingham, and Jacques Piette for advice;
Dr. Charlotte Sandersen for horse management; Renata Turlej for
manuscript correction; and Carine Gresse, Martine Leblond, Michel
Motkin, Jean-François Rouelle, and Ilham Sbaï for
excellent technical and secretarial assistance. |
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Fabrice Bureau, Department of Physiology, Faculty of Veterinary Medicine, University of Liege, Bâtiment B42, Sart Tilman, B-4000 Liege, Belgium.
3 Abbreviations used in this paper: BBS, bronchial brushing sample; HA, hemagglutinin; BEC, bronchial epithelial cell.
Received for publication February 22, 2000. Accepted for publication August 23, 2000.
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