 B,
Adhesion Molecules, and Cytokines in Asthma
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ABSTRACT |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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The asthmatic inflammatory response can be attenuated by corticosteroids and in part by 
2-agonists. We investigated if these effects are accompanied by a downregulation in nuclear factor kappa B (NF-
B), a transcription factor regulating many of the cytokine and adhesion molecule genes expressed in allergic inflammation. Bronchial biopsies were taken before and after 8 wk treatment with formoterol, budesonide, or placebo from atopic asthmatics. Biopsies were processed into glycol methacrylate and stained immunohistochemically for eosinophils (as an index of inflammation), activated and total NF-B, adhesion molecules, and cytokines. After budesonide treatment there was a significant decrease
in the number of submucosal cells staining for total NF-B, granulocyte macrophage colony-stimulating factor (GM-CSF) and tumor necrosis factor-alpha (TNF-
), accompanied by a significant
decrease in mucosal eosinophils and expression of vascular cell adhesion molecule-1 (VCAM-1) in the endothelium and interleukin-8
(IL-8) in the epithelium. After formoterol treatment there was a significant decrease in eosinophils and the epithelial expression of activated NF-B, but these changes were not accompanied by reduced
immunoreactivity for adhesion molecules or cytokines. We conclude
that at least some of the therapeutic efficacy of inhaled corticosteroids is mediated through inhibition of NF-B-regulated gene expression, whereas the reduction in airway eosinophilia by long-acting 2-agonists probably operates through alternative pathways.
Keywords: budesonide; formoterol; NF-B; immunohistochemistry;
asthma
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Corticosteroids, the mainstay controller drugs in asthma, dramatically reduce both the influx and residence time of eosinophils in the respiratory mucosa (1). This is paralleled by a
reduction in the transcription and expression of both leukoattractant and antiapoptotic cytokines (3) and adhesion molecules (5). The effects of 2-agonists on the underlying inflammation are less clear. The inhaled long-acting adrenoreceptor
2-agonist formoterol decreased eosinophil infiltration when
assessed in bronchial mucosal biopsies (6), but no effects on
inflammatory indices were observed in biopsies or bronchoalveolar lavage (BAL) after treatment with salmeterol (7, 8) or
in biopsies following terbutaline (9). Protection against allergen-induced inflammatory cell influx has been observed in salmeterol-treated asthmatics (10, 11).
Attention is now being focused on the mechanisms that
modulate these anti-inflammatory events at the gene level. Of
particular interest is the role of transcription factors. Nuclear
factor kappa B (NF-B) is potentially a key target on account
of its role in regulating the transcription of proinflammatory
genes known to be involved in the allergic tissue response (12,
13). These include genes encoding for intercellular adhesion
molecule-1 (ICAM-1), vascular cell adhesion molecule-1
(VCAM-1), and E selectin, and the cytokines granulocyte
macrophage colony-stimulating factor (GM-CSF), tumor necrosis factor- (TNF-), interleukin-6 (IL-6), and chemokines
belonging to both the C-C and C-X-C families (12, 13). NF-B
exists as an "inactive" form in the cytoplasm of cells because
of the reversible binding of an inhibitory protein, IB, which
prevents its translocation to the nucleus by overlying a nuclear
translocation site (NLS) (14). Activation of NF-B, after
cleavage of IB can be initiated by a range of stimuli, including TNF-, interleukin-1 (IL-1), interleukin-2 (IL-2), leukotriene B4 (LTB4), respiratory viruses, and exposure to reactive oxygen species (14), all of which have been implicated in
the inflammatory response of asthma (15). NF-B regulatory
elements have been shown to be present in the endothelium,
epithelium, T and B lymphocytes, and macrophages (14),
which to varying extents are all targets for the action of corticosteroids and 2-agonists.
Studies in vitro have shown that the activation of NF-B
can be effectively suppressed by corticosteroids. Dexamethasone inhibits the TNF--induced upregulation of NF-B in
both monocytic and bronchial epithelial cell lines (16, 17), human peripheral blood mononuclear cells (18), and human lung
stimulated ex vivo (19). Because NF-B is involved in the expression of a wide range of proinflammatory molecules implicated in the cellular and mediator events of asthma, it is a logical
molecular target for the therapeutic action of corticosteroids.
However, the mechanisms by which long-term treatment with
2-agonist may modulate the inflammatory response are not
known, although there is some in vitro evidence that suggests
NF-B pathways may be involved. Salbutamol has been shown
to inhibit TNF- release from human peripheral blood monocytes (20) and skin mast cells (21) which could, in turn, effect
NF-B activation. Fenoterol has also been shown to inhibit
the interferon-
-induced expression of ICAM-1 on primary human bronchial epithelial cells (22).
To test the hypothesis that the anti-inflammatory effects of
the corticosteroid budesonide and the long-acting 2-agonist formoterol are mediated through NF-B-regulated pathways,
we have further analyzed endobronchial biopsies taken in a
previous placebo-controlled study (6) for NF-B expression
and indices of airway inflammation using novel immunohistochemical techniques employing two antibodies directed to
"active form" (Mab 2C7) and "active/inactive form" (total)
(Mab G96) of NF-B (23). Mab 2C7 is directed to the NLS site
on p65 subunit of NF-B; therefore, this epitope on NF-B will
only be revealed after activation and cleavage of IB to unmask
the NLS site (23). Mab G96 is directed to an epitope close to
the carboxyl end of p65 and therefore detects both unactivated trimer and activated dimer of NF-B (23).
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METHODS |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Study Design and Subjects
This was a randomized, double-blind, placebo-controlled study of 10 to 12 wk duration, conducted in northern Sweden (6). There was a 2- to 4-wk screening period followed by a 9-wk treatment phase with either inhaled 24 µg formoterol, 400 µg budesonide twice a day, or matched placebo. Of the 64 subjects evaluated in the initial study (6), a subgroup of 30, 10 in each treatment group were evaluated in this study (Table 1). This was principally based on the availability of tissue, the 10 largest samples from each of the original groups being selected. However, with a sample size of 10 in each group and assuming that 80% of the patients on active treatment compared with 10% on placebo will improve, a two-sided Fisher exact test with a 0.05 significance level will have at least 80% power to detect a difference. This is an approximation of the power achieved, using a Mann-Whitney U test.
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Subjects underwent two fiberoptic bronchoscopies, as previously described (6), and according to the latest guidelines (24), one before and the other after completing the 8-wk treatment with either formoterol, budesonide, or placebo. Study medication was taken on the day of bronchoscopy as usual. Bronchial mucosal biopsies were taken and processed for immunohistochemical analysis. The study was approved by the ethics committee of the University of Umeå, Sweden and subjects gave their informed written consent.
Biopsy Processing and Immunohistochemical Analysis
Bronchial mucosal biopsies were fixed overnight at 
20° C in acetone
containing protease inhibitors and then processed into glycol methacrylate (GMA) resin (25). Two-micrometer sections were cut and
stained immunohistochemically using the streptavidin-biotin peroxidase detection system and monoclonal antibodies (Mabs) directed to
eosinophils as an index of inflammation, NF-B and its inhibitor IB, and NF-B-regulated adhesion molecules and cytokines (Table 2). Negative control sections were incubated with isotype-matched immunoglobulins.
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After staining, the number of EG2-, NF-B-, and cytokine-positive cells per mm
2 submucosa and number of NF-B and adhesion
molecule positively stained microvessels as a percentage of the total
vessel number (identified by staining with the endothelium marker
EN4) were enumerated. The percentage expression of NF-B and cytokines in the epithelium were assessed with the assistance of computerized image analysis (Improvision, Birmingham, UK).
Mucosal biopsy sections from five of the 30 subjects with high levels of NF-B expression at baseline were stained using a double immunohistochemical technique, as previously described (23), to identify which cells in the submucosa were immunoreactive for activated
NF-B (Table 2). The percentage of the activated NF-B-positive
cells for each cell type was calculated.
Statistical Analyses
Data were analyzed using the SPSS statistical package. Within each
group, pretreatment and posttreatment values were compared using
the Wilcoxon rank sign test. The percentage change for each parameter between the groups was compared using the Mann-Whitney U
test. The presence of correlations between the level of NF-B expression and that for adhesion molecules and cytokines was tested using
the Spearman rank correlation test.
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RESULTS |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Eosinophils
The number of infiltrating eosinophils in the submucosa, used
as an index of inflammation, decreased significantly after
budesonide treatment from 2.45 to 0 mm2 (p = 0.02) and after formoterol from a median of 21.12 to 3.9 mm2 (p = 0.005). These changes were significant when compared with placebo in both the budesonide- and formoterol-treated
groups (p = 0.04 and p = 0.02, respectively). No significant
within-group change was observed with placebo (Figure 1).
This confirms our previous findings (6).
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NF-B and IB
A within-treatment analysis showed that 8 wk of budesonide
produced a significant reduction in the number of submucosal
cells staining for total NF-B (G96) from a median of 5.99 to
1.19 cells mm2 (p = 0.01), when compared with the pretreatment value (Figure 2A). This change was significant when
compared with either placebo (p = 0.007) or formoterol (p = 0.004), where no overall change in the number of cells staining
for total NF-B (G96) occurred. In all treatment groups there
were no significant changes in the number of submucosal inflammatory cells expressing immunoreactivity with the 2C7
Mab to activated NF-B (data not shown).
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Inhaled budesonide produced a within-group decrease in
percentage epithelial expression of active NF-B (2C7), from
a median value of 4.69% to 0.05% (p = 0.06), which was not
observed in the formoterol or placebo group (Figure 2B). A
significant (p = 0.03) between-group change in the level of immunoreactivity for activated NF-B was seen in the formoterol group compared with the placebo group. There were no
within-group or between-group changes in the percentage of
the epithelium expressing immunoreactivity for total NF-B
(G96) (data not shown). There was no change in the endothelial expression of total or activated NF-B after either active
treatments or placebo.
Staining for IB, was minimal with a median of zero cells
being stained in the submucosa (range 0 to 14.7 mm2), and no
staining being evident in either the endothelium or epithelium. Positive staining was observed using the same antibody applied to nasal polyp tissue used as a positive control. Representative photographs showing staining for total NF-B
(G96), activated NF-B (2C7), and IB are shown in Figure 3.
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Adhesion Molecules
When compared with pretreatment, budesonide but not formoterol or placebo resulted in a significant (p = 0.03) decrease in the proportion of vessels expressing the adhesion molecule VCAM-1, from a median value of 22.56% to 4.52% (Figure 4). However, between-treatment comparisons did not achieve significance. There were no changes in endothelial expression of ICAM-1 and E selectin.
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Cytokines
In the budesonide but not in the formoterol or placebo group, there
was a significant decrease in the number of submucosal cells immunoreactive for GM-CSF, from a median of 6.67 to 0 cells mm2
(p = 0.01), and TNF- from a median of 4.84 to 0 cells mm2
(p = 0.01) (Figures 5A and 5B). In between-treatment comparisons, the budesonide-induced decrease in GM-CSF and
TNF- expression was significant when compared with placebo (p = 0.02 and p < 0.001, respectively), and the decreased
immunoreactivity for TNF- was also significant when compared with formoterol (p = 0.007).
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In the epithelium, a significant decrease in interleukin-8 (IL-8) immunoreactivity was observed in the budesonide-treated group (p = 0.05) and the placebo group (p = 0.03), from a median of 3.73% to 0.85% and a median of 7.49% to 2.51%, respectively (Figure 5C). These changes were not significant between treatment groups.
No significant within- or between-treatment group changes
were seen for IL-8 in the submucosa; IL-8, GM-CSF, and
TNF- in the endothelium; or GM-CSF and TNF- in the epithelium (data not shown).
Cellular Provenance of NF-B
Using appropriate cell-specific Mabs in the bronchial submucosa double staining revealed that activated NF-B (2C7) localized to mast cells, eosinophils, T and B lymphocytes, and
macrophages. Of the total number of submucosal cells immunoreactive for the activated form of NF-B, 46% were mast
cells, 9% eosinophils, 9% T lymphocytes, 25% B lymphocytes,
and 11% macrophages. Examples of double staining for NF-B
in mast cells and eosinophils are shown in Figure 6.
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Relationships between Inflammatory Indices
Several associations between NF-B, adhesion molecule, and
cytokine expression were observed; these are summarized in
Table 3.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Building on the knowledge that regular treatment with inhaled formoterol or budesonide causes a reduction in eosinophil infiltration into the bronchial submucosa (5), we have attempted to explore a possible mechanism involving the regulation
of the transcription factor NF-B.
We have observed the expression of NF-B in the epithelium and cells within the submucosa of asthmatics as previously described by Hart and coworkers (26), but in addition,
we have demonstrated its expression in the endothelium. Using Mab 2C7, we have also shown immunoreactivity for the
activated form of NF-B in the epithelium, endothelium, and
submucosal cells and have identified these submucosal cells as
mast cells, eosinophils, macrophages, and T and B lymphocytes.
Budesonide treatment resulted in a clear reduction in submucosal inflammatory cells immunostaining with the Mab directed to total but not to the activated form of NF-B, and this
was paralleled by a reduction in cells staining for both GM-CSF and TNF-, cytokines of critical importance to the airway
inflammatory response (15). In the endothelium, budesonide
reduced the proportion of submucosal vessels expressing
VCAM-1 and in the epithelium reduced IL-8 immunoreactivity was observed.
These observations, taken together with the correlation of
NF-B expression with that for cytokines and adhesion molecules, further support a role for NF-B in the regulation of the
allergic inflammatory response in asthma and suggest a molecular target through which glucocorticoids might be exerting at
least some of their therapeutic effects. A decrease in VCAM-1
expression, a vascular adhesion molecule specifically involved
in interactions with very late antigen-4 (VLA-4) expressed on
eosinophils, basophils, and lymphocytes (27) will result in reduced cell recruitment. TNF- has an important role in the
perpetuation of the allergic inflammatory response, inducing
the expression of VCAM-1 through the activation of NF-B
(13, 14). IL-8 is a potent chemoattractant for eosinophils
alone, but especially when associated with the secretory piece
of IgA (15). A reduction in IL-8 release from the epithelium
will most likely result in a decrease in the migration of eosinophils through the allergic mucosa to the airway lumen. The decrease in epithelial GM-CSF will affect the survival and activation of eosinophils present in the epithelium (15).
Our study supports previous in vitro studies (16) that
have demonstrated a decrease in NF-B activity after corticosteroid treatment. It also adds to the observation of Hancox
and coworkers (28), who in a small study of seven asthmatic
subjects, showed by gel shift assay applied to nuclear extracts
from bronchial biopsies that budesonide (but not terbutaline)
administered for 6 wk reduced NF-B DNA binding. However, the findings of both this study and that of Hancox and
coworkers (28) differ from the recent study by Hart and coworkers (29), who found that there was no change in NF-B
DNA binding and that an increase in epithelial nuclear expression of p65 occurred after steroid treatment. This discrepancy could be due to differences in the patients, treatment regimens, or in the histologic methods used to analyze the tissue
samples. The subjects in the study of Hart and coworkers (29)
were more hyperreactive than those in this study or that of
Hancox and coworkers (28) (PC20 methacholine of 0.45 mg/ml
versus 1.97 mg/ml and 1.37 mg/ml). In both our study and that
of Hancox and coworkers (28) budesonide was administered for 8 or 6 wk, whereas in that of Hart (29), fluticasone was given for only 4 wk. Also, in our study we used GMA-embedded samples and a specific, well-validated Mab directed to the
activated form of NF-B (23). This approach enabled us to
precisely localize immunoreactive signals in thin sections (2 µm)
with superior morphology over any other tissue processing
method (25). Hart and coworkers (29) used a polyclonal antibody directed to the p65 subunit of NF-B, that was not specific for identifying the activated form, and moreover, they applied this polyclonal antibody to frozen sections and relied on
cellular localization on thick sections (6 µm) of presumed
poor morphology, from which they inferred activation. We
firmly believe that GMA-embedded tissues that can be thin-sectioned, through individual cells, provide a more precise
method for the intracellular localization of immunoreactivity.
The molecular mechanisms through which glucocorticoids
exert their effects on NF-B-regulated genes are not well understood, with several possibilities being suggested. An indirect mechanism may be the increased production of the NF-B
inhibitor IB (30, 31), thereby leading to a decrease in active
NF-B dimers. Our study would not support this as the low
level of immunoreactive IB expression observed in the
asthmatic bronchial mucosa showed no evidence of increasing
after 8 wk of continuous budesonide treatment. Additionally,
in the A549 and H292 lung epithelial cell lines, an inhibition of
NF-B activation by dexamethasone is reported but without
an accompanying increase in IB (17, 18, 32).
A potential direct mechanism for the action of corticosteroids involves physical interaction of the glucocorticoid/receptor complex and NF-B (33, 34), which prevents NF-B binding
to DNA and inducing transcription. It is suggested that the
glucocorticoid/receptor complex (GC/GCR) binds to a mini-leucine zipper region near the carboxyl terminal end of the
p65 subunit (34). Our observations with the two different
monoclonal antibodies to NF-B would lend support to this
view, as does the decreased NF-B DNA binding reported by
Hancox and coworkers (28). We only observed a decrease in
NF-B immunoexpression with the Mab directed to the
epitope expressed on total NF-B (activated and unactivated)
(G96) and not that to the activated form of NF-B (2C7). Because the Mab G96 recognizes an epitope close to the carboxyl end of p65, this epitope would be obscured when the
steroid/GCR complex becomes bound to NF-B, causing a reduction in immunoreactivity. In contrast, the NLS in being located some distance from the C terminus would not be obscured and would still be available to interact with the Mab
2C7, resulting in little or no apparent change in the level of immunoreactivity seen.
Although in this study we did not observe a decrease in the
expression of total or activated NF-B after 8 wk treatment
with formoterol in the submucosa or endothelium, a significant decrease in expression of activated NF-B compared
with the placebo group was seen in the epithelium. No parallel
changes in the expression of the epithelial cytokines IL-8,
GM-CSF, or TNF- were observed. This suggests that other
NF-B-regulated genes may be involved.
In addition to testing our hypothesis regarding the involvement of NF-B pathways in the modulation of the inflammatory response in asthma by corticosteroids and 2-agonists, we
have gained other important data. Double immunohistochemical staining showed that mast cells were the principal cells expressing activated NF-B (46%), followed by B lymphocytes
(25%), T lymphocytes (10%), and macrophages (10%). The
expression of activated NF-B in B cells is an indicator that
these cells are actively involved in immunoglobulin synthesis
because these genes are closely regulated by NF-B (14). T
lymphocytes and macrophages have also been shown to contain NF-B regulatory elements in the promoter regions of
several cytokine genes relevant to the allergic inflammatory
response (13, 14). However, to our knowledge, this is the first
report indicating that human mast cells are a major source
NF-B in asthma. This supports our earlier work (35) which
has revealed that mast cells isolated from human lung contain
NF-B regulatory elements and that activation of NF-B can
be stimulated by TNF-- and IgE-dependent mechanisms. This
is accompanied by an increase in the expression of TNF-, IL-8,
and GM-CSF. These findings recognize the important role of
this transcription factor in regulating mast cell cytokines that
are implemented in the recruitment of leukocytes, including eosinophils, into the asthmatic airway (12).
Comparison of immunostaining at baseline by the two different NF-B antibodies in the submucosa, endothelium, and
epithelium revealed strong correlations. Despite being able to
demonstrate clear immunoreactivity for IB in nasal polyp
tissue in bronchial mucosa, fixed and embedded under identical conditions, the expression of this inhibitory component
was minimal. Taken together, these observations suggest that
the NF-B detected in the bronchial mucosa of asthmatic subjects was mostly in the active dimeric form, as has been reported recently by Hart and coworkers (26) using gel shift assays applied to bronchial biopsies and induced sputum.
In conclusion, we have shown that the inhaled corticosteroid, budesonide is effective in reducing the allergic inflammatory reaction in the bronchial mucosa of mild asthmatic subjects and that this may, in part, be mediated through a direct
interaction of budesonide with NF-B. Further studies will be
needed to corroborate these findings. We have confirmed the
anti-inflammatory effects of formoterol as previously reported
(6) but have not been able to clearly demonstrate that NF-B-mediated pathways are involved. We can also conclude that
the majority of NF-B in the bronchial mucosa is in an activated state and that mast cells are the principal inflammatory
cell containing activated NF-B.
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Footnotes |
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Correspondence and requests for reprints should be addressed to Dr. Susan J. Wilson, Medical Specialities (RCMB), Mailpoint 810, Centre Block, Level D, Southampton General Hospital, Tremona Road, Southampton SO16 6YD, U.K. E-mail: sjw1{at}soton.ac.uk
(Received in original form October 10, 2000 and in revised form February 26, 2001).
Acknowledgments: The authors thank Janet Underwood for assistance with immunohistochemical staining.
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References |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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