American Journal of Respiratory and Critical Care Medicine

The asthmatic inflammatory response can be attenuated by corticosteroids and in part by β2-agonists. We investigated if these effects are accompanied by a downregulation in nuclear factor kappa B (NF- κ 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- κ B, adhesion molecules, and cytokines. After budesonide treatment there was a significant decrease in the number of submucosal cells staining for total NF- κ B, granulocyte macrophage colony–stimulating factor (GM-CSF) and tumor necrosis factor-alpha (TNF- α ), 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- κ 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- κ B-regulated gene expression, whereas the reduction in airway eosinophilia by long-acting β2-agonists probably operates through alternative pathways.

Keywords: budesonide; formoterol; NF-κB; immunohistochemistry; asthma

Corticosteroids, the mainstay controller drugs in asthma, dramatically reduce both the influx and residence time of eosinophils in the respiratory mucosa (1-4). 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 β2-agonists on the underlying inflammation are less clear. The inhaled long-acting adrenoreceptor β2-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-κB) 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-α (TNF-α), interleukin-6 (IL-6), and chemokines belonging to both the C-C and C-X-C families (12, 13). NF-κB exists as an “inactive” form in the cytoplasm of cells because of the reversible binding of an inhibitory protein, IκB, which prevents its translocation to the nucleus by overlying a nuclear translocation site (NLS) (14). Activation of NF-κB, after cleavage of IκB can be initiated by a range of stimuli, including TNF-α, interleukin-1β (IL-1β), 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-κB 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 β2-agonists.

Studies in vitro have shown that the activation of NF-κB can be effectively suppressed by corticosteroids. Dexamethasone inhibits the TNF-α-induced upregulation of NF-κB 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-κB 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 β2-agonist may modulate the inflammatory response are not known, although there is some in vitro evidence that suggests NF-κB pathways may be involved. Salbutamol has been shown to inhibit TNF-α release from human peripheral blood monocytes (20) and skin mast cells (21) which could, in turn, effect NF-κB activation. Fenoterol has also been shown to inhibit the interferon-γ-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 β2-agonist formoterol are mediated through NF-κB-regulated pathways, we have further analyzed endobronchial biopsies taken in a previous placebo-controlled study (6) for NF-κB 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-κB (23). Mab 2C7 is directed to the NLS site on p65 subunit of NF-κB; therefore, this epitope on NF-κB will only be revealed after activation and cleavage of IκB 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-κB (23).

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.


Clinical Parameters* PlaceboBudesonideFormoterol
Age, yr28.8 ± 8.631.6 ± 8.026.9 ± 9.4
FEV1 Pretreatment, L 3.89 ± 1.01 3.70 ± 0.733.40 ± 0.72
 % predicted     91 ± 14      92 ± 10     94 ± 9
FEV1 Posttreatment, L3.66 ± 0.833.70 ± 0.803.40 ± 0.71
 % predicted     86 ± 11      92 ± 10     94 ± 9
PC20 Pretreatment,  methacholine mg/ml1.37 ± 2.111.79 ± 1.941.05 ± 2.34
PC20 Posttreatment,  methacholine mg/ml1.83 ± 4.96 2.43 ± 4.632.63 ± 4.07

*The mean ± 1SD is shown for age and FEV1, and geometric mean ± 1SD for PC20 methacholine.

Significant changes before and after treatment.

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-κB and its inhibitor IκBα, and NF-κB-regulated adhesion molecules and cytokines (Table 2). Negative control sections were incubated with isotype-matched immunoglobulins.


Eosinophil cationic proteinEG2Pharmacia & Upjohn, Milton Keynes, UK
NF-κB totalG96Pharmingen, San Diego, CA, USA
NF-κB2C7Pharmacia & Upjohn, Kalamazoo,  MI, USA (gift)
IκBαH4Santa Cruz, CA, USA
ICAM-1RR1Boehringer-Ingleheim, Ridgefield, CT (gift)
VCAM-11.4C3Serotec, Kidlington, UK
E selectin1.2B6Serotec, Kidlington, UK
IL-8NAP IIAlexis Corporation, Nottingham, UK
GM-CSFGenzyme, West Malling, UK
TNF-α52B83Celltech, Slough, UK (gift)
Mast cell tryptaseAA1Dako, Ely, UK
T lymphocytes–CD3UCHT1Dako, Ely, UK
B lymphocytes–CD20L26Dako, Ely, UK
Macrophages–CD14TÜK4Dako, Ely, UK

After staining, the number of EG2-, NF-κB-, and cytokine-positive cells per mm−2 submucosa and number of NF-κB 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-κB 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-κB 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-κB (Table 2). The percentage of the activated NF-κB-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-κB expression and that for adhesion molecules and cytokines was tested using the Spearman rank correlation test.


The number of infiltrating eosinophils in the submucosa, used as an index of inflammation, decreased significantly after budesonide treatment from 2.45 to 0 mm−2 (p = 0.02) and after formoterol from a median of 21.12 to 3.9 mm−2 (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).

NF- κ B and I κ B

A within-treatment analysis showed that 8 wk of budesonide produced a significant reduction in the number of submucosal cells staining for total NF-κB (G96) from a median of 5.99 to 1.19 cells mm−2 (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-κB (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-κB (data not shown).

Inhaled budesonide produced a within-group decrease in percentage epithelial expression of active NF-κB (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-κB 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-κB (G96) (data not shown). There was no change in the endothelial expression of total or activated NF-κB after either active treatments or placebo.

Staining for IκBα, was minimal with a median of zero cells being stained in the submucosa (range 0 to 14.7 mm−2), 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-κB (G96), activated NF-κB (2C7), and IκB are shown in Figure 3.

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.


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 mm−2 (p = 0.01), and TNF-α from a median of 4.84 to 0 cells mm−2 (p = 0.01) (Figures 5A and 5B). In between-treatment comparisons, the budesonide-induced decrease in GM-CSF and TNF-α expression was significant when compared with placebo (p = 0.02 and p < 0.001, respectively), and the decreased immunoreactivity for TNF-α was also significant when compared with formoterol (p = 0.007).

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-α in the endothelium; or GM-CSF and TNF-α in the epithelium (data not shown).

Cellular Provenance of NF- κ B

Using appropriate cell-specific Mabs in the bronchial submucosa double staining revealed that activated NF-κB (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-κB, 46% were mast cells, 9% eosinophils, 9% T lymphocytes, 25% B lymphocytes, and 11% macrophages. Examples of double staining for NF-κB in mast cells and eosinophils are shown in Figure 6.

Relationships between Inflammatory Indices

Several associations between NF-κB, adhesion molecule, and cytokine expression were observed; these are summarized in Table 3.


NF-κB total (G96)NF-κB active (2C7)
NF-κB active (2C7)Submucosa: r = 0.77, p < 0.001NA
Endothelium: r = 0.83, p < 0.001
Epithelium: r = 0.59, p = 0.001
VCAM-1Endothelium: r = 0.55, p = 0.002Endothelium: r = 0.48, p = 0.007
E selectinEndothelium: r = 0.39, p = 0.03Endothelium: r = 0.41, p = 0.03
GM-CSFSubmucosa: r = 0.52, p = 0.004Submucosa: r = 0.52, p = 0.003
Epithelium: r = 0.38, p = 0.04Epithelium: r = 0.72, p < 0.001
TNF-αSubmucosa: r = 0.68, p < 0.001 Submucosa: r = 0.72, p = 0.001
IL-8Epithelium: r = 0.42, p = 0.03No correlations

Definition of abbreviation: NA = not applicable.

*Determined by the Spearman rank test between the expression of NF-κB and adhesion molecules and cytokines in the bronchial mucosa.

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-κB.

We have observed the expression of NF-κB 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-κB 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-κB, and this was paralleled by a reduction in cells staining for both GM-CSF and TNF-α, 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-κB expression with that for cytokines and adhesion molecules, further support a role for NF-κB 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-α has an important role in the perpetuation of the allergic inflammatory response, inducing the expression of VCAM-1 through the activation of NF-κB (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 (16-19) that have demonstrated a decrease in NF-κB 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-κB 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-κB 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-κB (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-κB, 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-κB-regulated genes are not well understood, with several possibilities being suggested. An indirect mechanism may be the increased production of the NF-κB inhibitor IκBα (30, 31), thereby leading to a decrease in active NF-κB dimers. Our study would not support this as the low level of immunoreactive IκBα 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-κB activation by dexamethasone is reported but without an accompanying increase in IκBα (17, 18, 32).

A potential direct mechanism for the action of corticosteroids involves physical interaction of the glucocorticoid/receptor complex and NF-κB (33, 34), which prevents NF-κB 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-κB would lend support to this view, as does the decreased NF-κB DNA binding reported by Hancox and coworkers (28). We only observed a decrease in NF-κB immunoexpression with the Mab directed to the epitope expressed on total NF-κB (activated and unactivated) (G96) and not that to the activated form of NF-κB (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-κB, 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-κB after 8 wk treatment with formoterol in the submucosa or endothelium, a significant decrease in expression of activated NF-κB 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-α were observed. This suggests that other NF-κB-regulated genes may be involved.

In addition to testing our hypothesis regarding the involvement of NF-κB pathways in the modulation of the inflammatory response in asthma by corticosteroids and β2-agonists, we have gained other important data. Double immunohistochemical staining showed that mast cells were the principal cells expressing activated NF-κB (46%), followed by B lymphocytes (25%), T lymphocytes (10%), and macrophages (10%). The expression of activated NF-κB in B cells is an indicator that these cells are actively involved in immunoglobulin synthesis because these genes are closely regulated by NF-κB (14). T lymphocytes and macrophages have also been shown to contain NF-κB 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-κB in asthma. This supports our earlier work (35) which has revealed that mast cells isolated from human lung contain NF-κB regulatory elements and that activation of NF-κB can be stimulated by TNF-α- and IgE-dependent mechanisms. This is accompanied by an increase in the expression of TNF-α, 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 (12-14).

Comparison of immunostaining at baseline by the two different NF-κB antibodies in the submucosa, endothelium, and epithelium revealed strong correlations. Despite being able to demonstrate clear immunoreactivity for IκBα 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-κB 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-κB. 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-κB-mediated pathways are involved. We can also conclude that the majority of NF-κB in the bronchial mucosa is in an activated state and that mast cells are the principal inflammatory cell containing activated NF-κB.

The authors thank Janet Underwood for assistance with immunohistochemical staining.

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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:


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