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Published ahead of print on July 31, 2003, doi:10.1164/rccm.200205-479OC
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American Journal of Respiratory and Critical Care Medicine Vol 168. pp. 1190-1198, (2003)
© 2003 American Thoracic Society

Persistent Activation of Nuclear Factor–{kappa}B Signaling Pathway in Severe Uncontrolled Asthma

Rosalia Gagliardo, Pascal Chanez, Marc Mathieu, Andreina Bruno, Giorgia Costanzo, Claire Gougat, Isabelle Vachier, Jean Bousquet, Giovanni Bonsignore and Antonio M. Vignola

Istituto di Biomedicina e Immunologia Molecolare, Consiglio Nazionale delle Ricerche; Istituto di Medicina Generale e Pneumologia, Universita' degli Studi di Palermo, Palermo, Italy; and U454 and Service des Maladies Respiratoires, Institut National de la Santé et de la Recherche Médicale, CHU de Montpellier, Montpellier, France

Correspondence and requests for reprints should be addressed to Rosalia Gagliardo, Ph.D., Istituto di Biomedicina e Immunologia Molecolare, Consiglio Nazionale delle Ricerche, Palermo, Via Ugo La Malfa 153, 90146 Palermo, Italy. E-mail: gagliardo.r{at}iol.it


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The transcription factor nuclear factor–{kappa}B (NF-{kappa}B) is inactive when bound to its inhibitory protein I{kappa}B{alpha}. On cell stimulation with inflammatory signals, I{kappa}B{alpha} is phosphorylated by I{kappa}B kinases and subsequently degraded. Freed NF-{kappa}B then induces expression of cytokines such as granulocyte–macrophage colony-stimulating factor, interleukin-8, and regulated upon activation, normal T cell expressed and secreted. These mediators are overexpressed in asthma and are downregulated by glucocorticoids through NF-{kappa}B activity repression. However, high levels of granulocyte–macrophage colony-stimulating factor, interleukin-8, and regulated upon activation, normal T cell expressed and presumably secreted are released by peripheral blood mononuclear cells isolated from patients with severe asthma despite continuous systemic glucocorticoid treatment. We report that these mediators are markedly decreased by pyrrolidinedithiocarbamate, an inhibitor of NF-{kappa}B activation. To further characterize the persistent NF-{kappa}B activation in severe asthma, we analyzed the expression of various components of this activation pathway in healthy subjects and in asthmatics with mild controlled, and moderate and severe uncontrolled disease. We found high amounts of phosphorylated I{kappa}B{alpha} characterizing the three asthmatic groups. Western blot analyses indicated that in peripheral blood mononuclear cells the I{kappa}B kinase ß and p65 levels were greater in moderate and severe asthmatics than in normal subjects. Electrophoretic mobility shift assay and immunocytochemistry showed a greater activation status of p65 in severe asthmatics. Our data suggest that exaggerated NF-{kappa}B activation perpetuates inflammatory mediators production in severe asthma.

Key Words: severe asthma • inflammation • glucocorticoids • nuclear factor–{kappa}B

The transcription factor nuclear factor–{kappa}B (NF-{kappa}B) plays a central role in immune and inflammatory responses. Indeed, it induces expression of many inflammatory mediators and is itself activated by inflammatory stimuli (1). NF-{kappa}B is a dimer comprised of subunits that can include c-Rel, RelA (p65), RelB, p50, and p52. In most cells, the NF-{kappa}B prototype is a heterodimer composed of p65 and p50, the former subunit carrying the transactivating function (2). Endogenous inhibitors, known as I{kappa}B, tightly regulate NF-{kappa}B activation by complexing with the transcription factor and trapping it in the cytoplasm. I{kappa}B molecules form a distinct family of proteins that includes I{kappa}B{alpha}, I{kappa}Bß, I{kappa}B{epsilon}, I{kappa}B{phi}, I{kappa}B{delta}, Bcl3, and p105 (3). The most characterized NF-{kappa}B inhibitor is I{kappa}B{alpha}. This protein binds avidly to the p65 subunit of NF-{kappa}B through its ankyrin repeat domains that associate with the nuclear localization signal and the immunoglobulin-like domain of p65 (4).

During activation of NF-{kappa}B, numerous stimuli, including interleukin (IL)-1 and tumor necrosis factor–{alpha}, activate a complex of I{kappa}B kinases (IKK) that phosphorylate I{kappa}B{alpha} on the amino terminus at serine residues 32 and 36 (5). The IKK complex contains two catalytic subunits, IKK{alpha} and IKKß, both of which phosphorylate I{kappa}B, and a regulatory subunit, IKK{gamma}, that is postulated to serve as a recognition site for upstream activators (3). Phosphorylation of I{kappa}B{alpha} leads to its immediate polyubiquitinylation, which targets I{kappa}B{alpha} for rapid degradation by the proteasome. As a result, free NF-{kappa}B dimers translocate to the nucleus and activate transcription of target genes (reviewed in Rothwarf and Karin [3]).

Activation of NF-{kappa}B leads to an increased expression of granulocyte–macrophage colony-stimulating factor (GM-CSF) and IL-8. Expression of these cytokines is normally inhibited by glucocorticoids (GCs) through repression of NF-{kappa}B activity (6, 7). However, despite continuous long-term treatment with systemic and high doses of inhaled GC, high levels of GM-CSF and IL-8 are synthesized by airway cells and peripheral blood mononuclear cells (PBMC) of severe asthmatics (8–10). These data suggest a persistent activation of NF-{kappa}B in severe asthma.

To test this hypothesis, we first analyzed whether PBMC isolated from severe asthmatics would release an increased amount of another cytokine that is upregulated by NF-{kappa}B (11) and is involved in the pathophysiology of asthma (12), namely regulated upon activation, normal T cell expressed and secreted (RANTES). We then compared the release of RANTES by PBMC of severe asthmatics with that of two other groups of asthmatics: (1) a group of subjects with moderate asthma who were clinically uncontrolled despite the use of high-dose inhaled GC; and (2) a group of mild asthmatics, who were clinically controlled and took inhalations of short-acting ß2 agonists as needed but not of GC. Because a comparable increased release of RANTES was observed in both uncontrolled severe and moderate asthmatics, this latter group of subjects underwent a short course of oral GC (1 mg/kg prednisolone for 10 days) to study GC responsiveness using both clinical and biological parameters.

We also examined whether the in vitro production of GM-CSF, IL-8, and RANTES by these cells was sensitive to pyrrolidinedithiocarbamate (PDTC), an inhibitor of NF-{kappa}B activation (13). To determine at which step(s) the NF-{kappa}B activation pathway was affected, we evaluated the level of p65, I{kappa}B{alpha}, phosphorylated I{kappa}B{alpha} (p-I{kappa}B{alpha}), IKK{alpha}, and IKKß in PBMC of the four study groups. Finally, to better characterize the qualitative regulation of p65, in PBMC of the same patients, we analyzed its subcellular localization by immunocytochemistry and its activation status by electrophoretic mobility shift assay (EMSA).


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Patients
Thirty-four asthmatic subjects were selected according to the criteria of the American Thoracic Society (14). The first group consisted of six subjects with mild intermittent asthma who took inhalations of short-acting ß2 agonists as needed but not of GC and were defined as subjects with mild controlled asthma. The second group consisted of eight patients with moderate asthma receiving inhaled GCs, who were uncontrolled recently (four puffs of short-acting ß2 agonists a day and two awakenings due to asthma during the last week) but did not take any systemic GC before the study, and were defined as subjects with moderate uncontrolled asthma. To improve asthma control, these patients were subsequently treated with a short course of oral GC (1 mg/kg prednisolone for 10 days), which is a current European medical practice. The third group consisted of 20 patients with severe uncontrolled asthma who required daily a high dose of inhaled GC (3,000 µg of beclomethasone dipropionate equivalent), long-acting ß2 agonists (100 µg salmeterol), and oral GC (median oral prednisone dose: 40 mg/day, as reported in Table 1) . These patients were all considered as severe uncontrolled and GC dependent because attempts to wean them from the systemic treatment have always failed in the previous year. Despite this important treatment, they had daily symptoms of asthma during the week preceding the study and required at least two puffs a day of short-acting ß2 agonist. Their bone mineral density in lumbar spine and femoral neck was decreased, as assessed by absorptiometry (mean T score was, respectively, -1.29 and -1.72), suggesting that they complied with their oral GC treatment. None of the asthmatic subjects was a current smoker. Five of 34 patients with asthma were ex-smokers (< 5 packs/year), but all of them had stopped smoking for more than 1 year before the beginning of the study. No differences were found between non- and ex-smokers for the different parameters studied.


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  		TABLE 1. Subject characteristics

 
Six healthy subjects comprised the control group. The study was approved by the Ethics Committee of the Montpellier hospital, and written informed consent was obtained from all patients.

Antibodies
Anti–heat shock protein 90 antibody (Transduction Laboratories, Lexington, KY) was diluted 1:1,000. Other antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA) and diluted as follows: 1:100, anti-p65; 1:200, I{kappa}B{alpha}; 1:50, anti–p-I{kappa}B{alpha}; 1:100, anti-IKK{alpha}; and 1:100, anti-IKKß.

Isolation of PBMC
PBMC were isolated by Ficoll–Hypaque gradient centrifugation, as described previously (10).

Production of Cytokines by PBMC
Freshly isolated PBMC were diluted at a concentration of one million cells/ml in Roswell Park Memorial Institute 1640 medium containing 10% heat-inactivated fetal calf serum, 100 U/ml penicillin, 100 µg/ml streptomycin, and 2 mM glutamin. Cells were cultivated for 3, 12, and 24 hours in the absence or in the presence of dexamethasone (Sigma, St. Louis, MO) or pyrrolidinedithiocarbamic acid (PDTC) (Alexis Biochemicals, San Diego, CA), as indicated in the figure legends. Supernatants were then harvested to measure their content in IL-8, GM-CSF, and RANTES by quantitative sandwich enzyme immunoassays following the manufacturer's recommendations (R&D Systems, Oxon, UK).

Western Blotting
Cell extracts from A549 cells and freshly isolated PBMC were prepared as described previously (10). Forty micrograms of total protein was subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis on 4 to 12% gradient gels and blotted onto nitrocellulose membranes. These were blocked with phosphate-buffered saline (PBS) containing 3% bovine serum albumin and 0.1% Tween 20 and then probed with primary antibodies. After serial washes with PBS containing 0.1% Tween 20, membranes were incubated with appropriate peroxidase-conjugated secondary antibodies. Blots were systematically probed with an anti–heat shock protein 90 antibody to ensure that equal amounts of cellular proteins were loaded per lane. Heat shock protein 90 is known to be abundantly expressed in various cell types (15). On each gel, 5 µg of total protein from A549 cells was loaded. For between-group comparisons, results were normalized and expressed as the ratio of the amount of p65, I{kappa}B{alpha}, p-I{kappa}B{alpha}, IKK{alpha}, and IKK{kappa}ß in PBMC relatively to that in A549 cells (external control), after correction with the density of the band obtained for heat shock protein 90 (internal control) for each patient.

Revelation was performed with an enhanced chemiluminescence system (Ambion, Austin, TX) followed by autoradiography. Films were analyzed by densitometric scanning using a monochrome charge-coupled device camera RS-170 (COHU, San Diego, CA) coupled to the NIH Image analysis software (NIH, Bethesda, MD).

Immunocytochemistry Analysis
Cytocentrifuge preparations of PBMC obtained from 6 of 6 normal subjects, 6 of 6 subjects with mild controlled asthma, 6 of 8 subjects with moderate uncontrolled asthma, and 6 of 20 subjects with severe uncontrolled asthma were made and fixed in cold acetone–chloroform (1:1). NF-{kappa}B immunoreactivity was evaluated using a goat polyclonal anti-p65 antibody (1:20). In control slides, antibody diluent (cod S0809; Dako, Glostrup, Denmark) was used according to the manufacturer's instructions. Revelation was performed through use of the labeled streptavidin–biotin method (Alkaline phosphatase Rabbit/Mouse/Goat; Universal LSAB+ Kit; Dako). p65 localization was expressed as percentage of PBMC showing a p65 nuclear staining.

Electrophoretic Mobility Shift Assay
Nuclear proteins were extracted from PBMC obtained from 6 of 6 normal subjects, 6 of 6 subjects with mild controlled asthma, 6 of 8 subjects with moderate uncontrolled asthma, and 6 of 20 subjects with severe uncontrolled asthma, according to the method of Schreiber and coworkers (16). The samples were resuspended in Buffer A (10 mM N-2-hydroxyethylpiperazine-N'-ethane sulfonic acid, pH 7.9, 10 mM KCl, 0.1 mM ethyleneglycol-bis-(ß-aminoethyl ether)-N,N'-tetraacetic acid, 0.1 mM ethylenediaminetetraacetic acid, 1 mM dithiothreitol, and 0.5 mM phenylmethylsulfonylfluoride [PMSF]). Cell lysis was induced with Nonidet P-40. After centrifugation at 12,000 rpm for 30 seconds at 4°C, the nuclear pellet was lysed with Buffer C (20 mM N-2-hydroxyethylpiperazine-N'-ethane sulfonic acid, pH 7.9, 0.4 M NaCl, 1 mM ethylenediaminetetraacetic acid, 1 mM ethyleneglycol-bis-(ß-aminoethyl ether)-N,N'-tetraacetic acid, 1 mM dithiothreitol, and 1 mM PMSF) and incubated on ice for 15 minutes. After brief centrifugation, the subsequent soluble fraction was stored at -80°C. To minimize proteolysis, all buffers contained a protease inhibitor cocktail (Roche, Mannheim, Germany). Mobility shift assays were performed as described originally by Dignam and coworkers (17). Double-stranded oligonucleotides encoding the consensus target sequence of NF-{kappa}B p65 subunit (Geneka Biotechnology, Inc., Montreal, PQ, Canada) were end-labeled using {gamma}-32P–adenosine triphosphate and T4 polynucleotide kinase (Invitrogen, Paisley, UK): 5'-AGC TTG GGG TAT TTC CAG CCG-3'; 3'-TCG AAC CCC ATA AAG GTC GGC-5'.

A double-stranded mutant oligonucleotide was used in competition assays to determine the protein specificity of the assay (Geneka Biotechnology, Inc.): 5'-AGC TTG GCA TAG GTC CAG CCG-3'; 3'-TCG AAC CGT ATC CAG GTC GGC-5'.

Nuclear extracts (5 µg) were incubated with 0.0175 pmol of 32P-labeled NF-{kappa}B oligonucleotide in binding reaction mixture (20% Ficoll, 175 mM NaCl, 300 mM KCl, 0.05% Nonidet P-40, pH 7.0). Nuclear extract derived from TPA+Cl-treated Jurkat cells was used as positive control (Geneka Biotechnology, Inc.). A 6% nondenaturing polyacrylamide gel was used for electrophoretic separation. DNA probes bound to NF-{kappa}B were retarded, whereas unbound (free) DNA probes migrated to the bottom of the electropherogram. Gels were vacuum dried and autoradiographed at -80°C using Kodak X-OMAT film.

Study Design
We measured RANTES release by PBMC isolated from 6 of 6 normal subjects, 6 of 6 subjects with mild controlled asthma, 8 of 8 subjects with moderate uncontrolled asthma, and 7 of 20 subjects with severe uncontrolled asthma. We examined the in vitro effect of dexamethasone on RANTES production by PBMC isolated from 8 of 8 subjects with moderate uncontrolled asthma and 7 of 20 subjects with severe uncontrolled asthma. We also tested the effect of PDTC, an inhibitor of NF-{kappa}B activation, on the production of IL-8, GM-CSF, and RANTES by PBMC obtained from 6 of 20 subjects with severe uncontrolled asthma.

Using Western blot analyses, we assessed the relative amount of the NF-{kappa}B p65 subunit, I{kappa}B{alpha}, p-I{kappa}B{alpha}, IKK{alpha}, and IKKß in PBMC isolated from 6 of 6 normal subjects, 6 of 6 subjects with mild controlled asthma, 8 of 8 subjects with moderate uncontrolled asthma, and 14 of 20 subjects with severe uncontrolled asthma. We then determined the level of p65, I{kappa}B{alpha}, p-I{kappa}B{alpha}, IKK{alpha}, and IKKß in PBMC isolated from eight of eight subjects with moderate uncontrolled asthma before and after 10 days of treatment with an oral dose of 1 mg/kg prednisolone. The effect of this treatment on RANTES production was also investigated.

To evaluate the p65 DNA binding, EMSA was performed using nuclear extract obtained from PBMC of 6 of 6 normal subjects, 6 of 6 subjects with mild controlled asthma, 6 of 8 subjects with moderate uncontrolled asthma before and after oral GC treatment, and 6 of 20 subjects with severe uncontrolled asthma.

Finally, to better characterize the qualitative regulation of p65 NF-{kappa}B subunit we analyzed, by immunocytochemistry, its subcellular localization in PBMC isolated from 6 of 6 normal subjects, 6 of 6 subjects with mild controlled asthma, 6 of 8 subjects with moderate uncontrolled asthma before and after oral GC treatment, and 6 of 20 subjects with severe uncontrolled asthma.

Some assays were performed on group subsets owing to limitation in the amount of cells recovered from certain subjects.

Statistical Analysis
Kruskal–Wallis and the Dunn's post tests were used for between-group comparisons. Mann–Whitney test was used for unpaired comparisons. Wilcoxon test was used for paired comparisons. Correlations were analyzed by Spearman rank test. Statistical significance was set at p < 0.05.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Characteristics of the Patients
Demographic characteristics of normal subjects and of mild controlled, moderate uncontrolled, and subjects with severe uncontrolled asthma are shown in Table 1. Subjects with severe uncontrolled asthma were older than the other subjects, but a significant difference in age was found only with subjects with mild controlled asthma (p < 0.001; Kruskal–Wallis and Dunn's post tests). Severe uncontrolled patients had asthma of a longer duration, but difference with the other groups of subjects was not significant. Since the year preceding the study, their median oral GC requirement was 40 mg/day, and 7 of 14 severe asthmatics were treated with a daily dose of 1 mg/kg. Airflow impairment remained high in subjects with severe uncontrolled asthma despite their chronic GC treatment and was similar to that in moderate uncontrolled patients.

The eight patients with moderate asthma who were uncontrolled recently received a short course of oral GC (1 mg/kg of prednisolone for 10 days). They were followed carefully, being called every day and attending our day hospital every 2 days, which is a way to improve compliance to oral GC and can be considered a directly observed treatment. Furthermore, all moderate asthmatics responded to the oral GC trial because all of them experienced an improvement in FEV1 (median, 25–75 percentiles before treatment: 58, 56.5–63.5; median, 25–75 percentiles after treatment: 89, 82–105; p = 0.0078; Wilcoxon test).

RANTES Production
We measured the spontaneous release of RANTES by PBMC isolated from six normal subjects, six subjects with mild controlled asthma, eight subjects with moderate uncontrolled asthma, and seven subjects with severe uncontrolled asthma. Cells were cultivated for 3, 12, and 24 hours. At any time point, the amount of RANTES released by PBMC of subjects with severe uncontrolled asthma and subjects with moderate uncontrolled asthma was higher than that of normal subjects (p < 0.01 and p < 0.05, respectively; Kruskal–Wallis and Dunn's post tests) (Figure 1A) . No significant correlation was found in severe asthmatics between the daily dose of oral GC and the levels of RANTES (r = 0.31; p = 0.4).



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  		Figure 1. Release of regulated upon activation, normal T cell expressed and secreted (RANTES) by peripheral blood mononuclear cells (PBMC). (A) Concentrations of RANTES released by PBMC isolated from six normal subjects (open squares), six subjects with mild controlled asthma (open circles), eight subjects with moderate uncontrolled asthma (closed circles), and seven subjects with severe uncontrolled asthma (open triangles). Individual data are shown. (B) Effect of a short-course oral glucocorticoid (GC) treatment on RANTES production. Concentrations of RANTES released by PBMC of subjects with moderate uncontrolled asthma (n = 8) before and after a short-course oral GC treatment (prednisolone at 1 mg/kg for 10 days). Individual data are shown. (C) and (D) Effect of in vitro dexamethasone (DEX) treatment on RANTES release. Concentrations of RANTES produced by PBMC isolated from subjects with moderate uncontrolled asthma (n = 8) and subjects with severe uncontrolled asthma (n = 7) and cultivated in the absence or in the presence of 0.1 µM DEX for 24 hours. Individual data are shown. Horizontal lines represent median values.

 
The amount of RANTES produced by PBMC of subjects with moderate uncontrolled asthma was markedly reduced after a short course oral GC treatment (p = 0.0078; Wilcoxon test) (Figure 1B).

When PBMC of moderate and subjects with severe uncontrolled asthma were cultivated in the presence of dexamethasone for 24 hours, RANTES release was inhibited by 71 and 72% (p < 0.0001 and p = 0.0006, respectively; Mann–Whitney test) (Figures 1C and 1D). Similar results were obtained at 3 and 12 hours and with PBMC isolated from mild asthmatics (data not shown).

Release of Cytokines after PDTC Treatment
PBMC isolated from subjects with severe uncontrolled asthma release high levels of IL-8, GM-CSF (10), and RANTES (Figure 1A). We measured the amount of IL-8, GM-CSF, and RANTES released by PBMC of 6 subjects with severe uncontrolled asthma and cultured for 24 hours in the absence or in the presence of PDTC, an inhibitor of NF-{kappa}B activation. PDTC treatment inhibited the release of IL-8, GM-CSF, and RANTES by 50, 34, and 32%, respectively (p < 0.01; Mann–Whitney test) (Figure 2) .



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  		Figure 2. Effect of in vitro pyrrolidinedithiocarbamate (PDTC) treatment on interleukin (IL)-8, granulocyte–macrophage colony-stimulating factor (GM-CSF), and RANTES release. Concentrations of (A) IL-8, (B) GM-CSF, and (C) RANTES produced by PBMC isolated from subjects with severe uncontrolled asthma (n = 6) and cultivated for 24 hours in the absence or in the presence of 500 µM PDTC. Individual data are shown. Horizontal lines represent the medians.

 
Protein Levels of the NF-{kappa}B p65 Subunit, I{kappa}B{alpha}, and p-I{kappa}B{alpha}
Expression of the NF-{kappa}B p65 subunit, I{kappa}B{alpha}, and p-I{kappa}B{alpha} in PBMC was investigated by Western blot analyses in 6 normal subjects, 6 subjects with mild controlled asthma, 8 subjects with moderate uncontrolled asthma, and 14 subjects with severe uncontrolled asthma. The relative amount of p65 in severe and subjects with moderate uncontrolled asthma was twofold higher than that in normal subjects (p < 0.01 and p < 0.05, respectively; Kruskal–Wallis and Dunn's post tests). Increased levels of p65 were also found in PBMC from mild controlled as compared with normal subjects, but differences did not reach significance (Figure 3) .



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  		Figure 3. Expression of p65 in PBMC of normal subjects and subjects with mild controlled, moderate uncontrolled, and severe uncontrolled asthma. (A) Representative Western blot analysis of p65 in PBMC from one normal subject (lane 2), two subjects with mild controlled asthma (lanes 3 and 4), two subjects with moderate uncontrolled asthma (lanes 5 and 6), and three subjects with severe uncontrolled asthma (lanes 7, 8, and 9). In lane 1, 5 µg of total protein from A549 cells was loaded on the gel as reference for p65 expression. (B) The same blot was probed with a monoclonal antibody directed against heat shock protein 90 (Hsp90). (C) Signals corresponding to p65 on the various Western blots were semiquantified by densitometric scanning, normalized, and expressed as the ratio of the amount of p65 in PBMC relatively to that in A549 cells (external control) after correction with the density of the band obtained for Hsp90 (internal control) for each patient. Horizontal lines represent the medians.

 
The three groups of patients with asthma were characterized by a much higher level of p-I{kappa}B{alpha} as compared with normal subjects (severe asthma: p < 0.001; moderate and mild asthma: p < 0.05; Kruskal–Wallis and Dunn's post tests) (Figures 4A and 4C) . In contrast, no overall difference was noted in the total amount of I{kappa}B{alpha} among the four groups. In each group, expression of I{kappa}B{alpha} was very variable, being undetectable in certain samples (Figure 4D). Nevertheless, in these latter samples, p-I{kappa}B{alpha} was usually detected, suggesting that the anti–p-I{kappa}B{alpha} antibodies were more sensitive than those recognizing total I{kappa}B{alpha}. Of note, we observed that the total amount of I{kappa}B{alpha} correlated with the level of p-I{kappa}B{alpha} in patients with asthma (mild controlled: r = 0.94, p = 0.03; moderate uncontrolled: r = 0.94, p < 0.01; severe uncontrolled: r = 0.94, p < 0.01; Spearman rank test) but not in normal subjects (r = -0.30, p = 0.7; Spearman rank test).



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  		Figure 4. Expression of phosphorylated I{kappa}B{alpha} (p-I{kappa}B{alpha}) and I{kappa}B{alpha} in PBMC of normal subjects and subjects with mild controlled, moderate uncontrolled, and severe uncontrolled asthma. (A) Representative Western blot analysis of p-I{kappa}B{alpha} in PBMC from one normal subject (lane 2), two subjects with mild controlled asthma (lanes 3 and 4), two subjects with moderate uncontrolled asthma (lanes 5 and 6), and three subjects with severe uncontrolled asthma (lanes 7, 8, and 9). In lane 1, 5 µg of total protein from A549 cells was loaded on the gel as reference for p-I{kappa}B{alpha} expression. (B) The same blot was probed with a monoclonal antibody directed against Hsp90. (C) and (D) Signals corresponding to p-I{kappa}B{alpha} and I{kappa}B{alpha} on the various Western blots were semiquantified by densitometric scanning, normalized, and expressed as the ratio of the amount of p-I{kappa}B{alpha} and I{kappa}B{alpha} in PBMC relatively to that in A549 cells (external control) after correction with the density of the band obtained for Hsp90 (internal control) for each patient. Horizontal lines represent the medians.

 
A short course of oral GC downregulated p65 by 20% (p < 0.01; Wilcoxon test) and p-I{kappa}B{alpha} by 40% (p < 0.01; Wilcoxon test) in PBMC of subjects with moderate uncontrolled asthma (Figure 6).



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  		Figure 6. Effect of a short-course oral glucocorticoid (GC) treatment on p65, phosphorylated I{kappa}B{alpha} (p-I{kappa}B{alpha}), and IKKß protein levels. Representative Western blot analysis of p65 (A), p-I{kappa}B{alpha} (D), IKKß (G), and Hsp90 (B, E, and H) in PBMC of a moderate uncontrolled asthmatic before and after oral GC treatment (prednisolone at 1 mg/kg for 10 days). Signals corresponding to p65 (C), p-I{kappa}B{alpha} (F), and IKKß (I) on the various Western blots were semiquantified by densitometric scanning, normalized, and expressed as the ratio of the amount of p65, p-I{kappa}B{alpha}, and IKKß in PBMC relatively to that in A549 cells (external control) after correction with the density of the band obtained for Hsp90 (internal control) for each patient.

 
Finally, in severe asthmatics, no significant correlation was found between the daily dose of oral GC and the levels of p65 subunit and p-I{kappa}B{alpha} (r = -0.17, p = 0.6; r = 0.09, p = 0.6, respectively).

IKK{alpha} and IKKß Protein Levels
Expression of IKK{alpha} and IKKß in PBMC was investigated, by Western blot analyses, in 6 normal subjects, 6 subjects with mild controlled asthma, 8 subjects with moderate uncontrolled asthma, and 14 subjects with severe uncontrolled asthma. The amount of IKKß in moderate and subjects with severe uncontrolled asthma was twofold higher than that in normal subjects (p < 0.05 and p < 0.01, respectively; Kruskal–Wallis and Dunn's post tests) (Figure 5) . Higher levels of IKKß protein were also expressed by PBMC from mild as compared with normal subjects, but differences were not statistically significant. In contrast, similar amounts of IKK{alpha} were found in PBMC isolated from the four study groups (data not shown). A short course of oral GC downregulated IKKß in PBMC of subjects with moderate uncontrolled asthma (p < 0.03; Figure 6) , whereas IKK{alpha} levels were not affected by this treatment (data not shown).



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  		Figure 5. Expression of I{kappa}B kinases (IKKß) in PBMC of normal subjects and subjects with mild controlled, moderate uncontrolled, and severe uncontrolled asthma. (A) Representative Western blot analysis of IKKß in PBMC from one normal subject (lane 2), two subjects with mild controlled asthma (lanes 3 and 4), two subjects with moderate uncontrolled asthma (lanes 5 and 6), and three subjects with severe uncontrolled asthma (lanes 7, 8, and 9). In lane 1, 5 µg of total protein from A549 cells was loaded on the gel as reference for IKKß expression. (B) The same blot was probed with a monoclonal antibody directed against Hsp90. (C) Signals corresponding to IKKß on the various Western blots were semiquantified by densitometric scanning, normalized, and expressed as the ratio of the amount of IKKß in PBMC relatively to that in A549 cells (external control) after correction with the density of the band obtained for Hsp90 (internal control) for each patient. Horizontal lines represent the medians.

 
Finally, in severe asthmatics no significant correlation was found between the daily dose of oral GC and the levels of IKK{alpha} and IKKß (r = 0.4 and p = 0.09, respectively).

Subcellular Distribution of p65 NF-{kappa}B Subunit
To determine the subcellular distribution of p65 NF-{kappa}B subunit, we performed immunocytochemistry analysis in PBMC isolated from six normal subjects, six subjects with mild controlled asthma, six subjects with moderate uncontrolled asthma, and six subjects with severe uncontrolled asthma.

Immunostaining showed that the percentage of PBMC with a p65 nuclear staining was higher in severe (median, 25–75 percentiles: 48, 45–50) and moderate asthmatics (median, 25–75 percentiles: 40, 35–45) than in mild asthmatics (median, 25–75 percentiles: 9.5, 8–10), and normal subjects (median, 25–75 percentiles: 0, 0–0.5) (p < 0.001) (Figure 7) .



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  		Figure 7. Subcellular distribution of p65 nuclear factor–{kappa}B (NF-{kappa}B) subunit by immunocytochemistry: p65 was localized in the cytosol of PBMC isolated from normal subjects (B), both in cytosol and nucleus in mild asthmatics (C), and mainly in the nucleus in moderate and subjects with severe uncontrolled asthma (D) and (E). Panels (E) and (F) show immunoreactivity for p65 in moderate asthmatics before and after the short course of oral GC. (A) Negative control (see METHODS).

 
Moreover, in moderate asthmatics, a short-course of oral GC reduced the percentage of PBMC with p65 nuclear immunoreactivity from 40 (32.5–43) to 12 (10.5–13.5) (p = 0.02) (Figures 7E and 7F).

NF-{kappa}B DNA Binding
Nf-{kappa}B p65 subunit activity was investigated, by EMSA analyses, in nuclear extract obtained from six normal subjects, six subjects with mild controlled asthma, six subjects with moderate uncontrolled asthma, and six subjects with severe uncontrolled asthma. EMSA was performed using specific 32P-labeled consensus target sequence of p65 subunit.

We found that none of the six normal subjects in whom EMSA was performed showed the formation of protein oligonucleotide complex. In addition, only three of six mild asthmatics showed p65 DNA binding, whereas all moderate and severe asthmatics showed a similar p65 DNA binding activity (Figure 8 , panel A).



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  		Figure 8. Electrophoretic mobility shift assay (EMSA) for the detection of specific oligonucleotide binding of activated p65 NF-{kappa}B subunit. Panel A: representative EMSA of nuclear proteins extracted from a normal subject (N), two subjects with mild controlled asthma (MC), three subjects with moderate uncontrolled asthma (MU), two subjects with severe uncontrolled asthma (SU); in first lane of panel A, EMSA of nuclear proteins extracted from Jurkat cells (positive control). Panel B: two representative EMSA of nuclear proteins extracted from moderate uncontrolled asthmatic before and after a short-course GC treatment. Panel C: the specificity of the p65 oligonucleotide binding was demonstrated in nuclear proteins extracted from Jurkat cells by the presence of the complex formation after the addition of a 100-fold molar excess of unlabeled mutant p65, used to compete with the labeled p65 probe. The complex formation was inhibited in the presence of an excess of unlabeled wild-type p65 oligonucleotide.

 
A short course of oral GC substantially inhibited the p65–oligonucleotide complex formation in PBMC of all subjects with moderate uncontrolled asthma (Figure 8, panel B).

Nuclear extracts derived from TPA+Cl-treated Jurkat cells were used in EMSA for specific mobility shift of p65-radiolabeled, double-stranded oligonucleotide (Figure 8, panel C). The complex formation was inhibited in the presence of an excess of unlabeled wild-type p65 oligonucleotide. The specificity of the p65 oligonucleotide binding was demonstrated by the presence of the complex formation after the addition of a 100-fold molar excess of unlabeled mutant p65 used to compete with the labeled p65 probe (Figure 8, panel C).


   DISCUSSION
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Despite maintenance treatment with systemic and inhaled GC, subjects with severe uncontrolled asthma present an ongoing inflammation of the airways characterized by an increased number of neutrophils (18, 19), activated T lymphocytes (20), and eosinophils (9, 21). This inflammation is probably sustained by cytokines like GM-CSF and IL-8 whose expression is increased not only in airway cells but also in PBMC isolated from subjects with severe uncontrolled asthma (9, 10, 22). We have shown previously that the persistent release of these inflammatory cytokines by PBMC of subjects with severe uncontrolled asthma is not due to abnormal expression of GC receptor–{alpha} or –ß (10), suggesting that other mechanisms may underlie the reduced responsiveness to GC. In this article, we report that, despite the long-term treatment with GC, PBMC of subjects with severe uncontrolled asthma spontaneously release high levels of RANTES, whose levels appeared to be high and heterogeneous, possibly reflecting more the clinical and biological heterogeneity characterizing severe asthma (18) than the different dose of oral steroid received. This latter interpretation of the results is supported by the lack of correlation between the doses of corticosteroids and the release of RANTES. We also show that severe uncontrolled asthma is characterized by an in vivo reduced responsiveness to GC, probably due to an inflammatory microenvironment sustained by a persistent NF-{kappa}B activation and release of mediators. It also appears that such a microenvironment is lost in vitro, leading to a dichotomy between ex vivo and in vitro responsiveness to corticosteroids, as shown by the inhibitory effect of exogenous dexamethasone on RANTES release. Moreover, in severe asthmatics, persistent NF-{kappa}B activation was also demonstrated by the increased p65 DNA binding, nuclear p65 localization, and increased level of p-I{kappa}B{alpha} and IKKß.

Although we acknowledge that the biological and clinical relevance of these results needs to be further supported by larger studies, these data extend previous observations showing that p65 expression is increased in bronchial epithelial cells of severe GC-dependent asthmatics (23). Importantly, p65 overexpression augments NF-{kappa}B transcriptional activity, as shown by transfection studies (24, 25). On cell stimulation with inflammatory mediators, I{kappa}B{alpha} is rapidly phosphorylated and proteolyzed, resulting in the liberation of NF-{kappa}B dimers that translocate into the nucleus (26, 27), activating the transcription of target genes among which are those of I{kappa}B{alpha} (27, 28). Thus, newly synthesized I{kappa}B{alpha} feedback inhibits NF-{kappa}B in the absence of a new signal (26, 27, 29, 30). We found that the level of I{kappa}B{alpha} correlated with that of p-I{kappa}B{alpha} in patients with asthma and that the three groups of patients with asthma harbored an overall much higher level of p-I{kappa}B{alpha} as compared with normal subjects. These observations suggest that resynthesized I{kappa}B{alpha} is immediately phosphorylated by incoming inflammatory stimuli and targeted for degradation. Therefore, constant phosphorylation of I{kappa}B{alpha} may prevent efficient negative feedback inactivation of NF-{kappa}B in PBMC of patients with asthma and may represent a marker of basal inflammation in asthma. Persistent activation of the NF-{kappa}B system seems to be associated with bad control of the disease in moderate and severe uncontrolled asthma. Possibly, in subjects with severe uncontrolled asthma, the excess of active NF-{kappa}B titrates GC receptor–{alpha}, thus impairing the antiinflammatory action of GC.

Because NF-{kappa}B induces expression of many inflammatory mediators and is itself activated by inflammatory signals (1), it is reasonable to believe that persistent NF-{kappa}B activation in severe uncontrolled asthma is caused by a strong inflammatory environment and vice versa. This implies that NF-{kappa}B activation would become sensitive to GC when the cells are taken away from their in vivo environment. We tested this hypothesis and found that the increased production of IL-8, GM-CSF, and RANTES by PBMC isolated from subjects with severe uncontrolled asthma was significantly reduced after in vitro addition of either GC or the specific NF-{kappa}B inhibitor PDTC (Ref. [10] and this study).

Increased activation of NF-{kappa}B was previously found in airway epithelial cells of mild asthmatics using EMSA and immunohistochemical examination (31). It has been reported that NF-{kappa}B DNA binding or p65 immunoreactivity was reduced in bronchial mucosa of mild or moderate asthmatics after several weeks of inhaled GC treatment (32, 33). We provide in this study evidence that GC reduce activation of NF-{kappa}B in PBMC of subjects with moderate uncontrolled asthma because an oral course of GC significantly decreased phosphorylation of I{kappa}B{alpha}, a crucial event in NF-{kappa}B activation, reduced nuclear p65 localization, and substantially inhibited the p65–DNA complex formation. Interestingly, all these events were not observed in severe uncontrolled asthma, despite the long-term GC treatment. Although it may be argued that the persistent activation of the NF-{kappa}B system in severe asthmatics is due to a suboptimal treatment with oral glucocorticosteroids, this seems to be unlikely because the activation of the NF-{kappa}B system was found even in subjects taking a dose of GC equivalent (1 mg/kg) to that used in uncontrolled moderate asthmatics during the steroid trial period. This issue is also supported by the lack of a correlation between the daily dose of oral GC and the levels of the different biomarkers studied. In addition, evaluation of prednisolone pharmacokinetics was performed on a randomized subgroup of patients with severe uncontrolled asthma (data not shown), and prednisolone clearance was within the expected range of normal values according to pharmacokinetic parameters of prednisolone after oral administration once daily (34).

IKK{alpha} and IKKß phosphorylate I{kappa}B{alpha} on specific serine residues, thus targeting I{kappa}B{alpha} for degradation and activating NF-{kappa}B (5). Gene knockout studies in mice indicate that IKKß is primarily responsible for the phosphorylation of I{kappa}B{alpha} in response to proinflammatory stimuli, whereas IKK{alpha} is essential for keratinocyte differentiation (35–40). We report that IKK{alpha} was expressed at similar levels in the four study groups. In contrast, the amount of IKKß in severe and subjects with moderate uncontrolled asthma was higher than that in normal subjects, suggesting a higher susceptibility of these patients to proinflammatory stimuli. This finding is in keeping with the increased release of RANTES by PBMC of the same study groups and indicates that the enhanced expression of this kinase can be associated with the ongoing inflammatory process and loss of asthma control in both moderate untreated asthmatics and severe asthmatics long-term treated with oral GC.

In conclusion, increased levels of activated p65, p-I{kappa}B{alpha}, and IKKß are found in PBMC of subjects with severe uncontrolled asthma. Our data suggest that NF-{kappa}B activation sustains the ongoing production of inflammatory mediators in these patients. We speculate that the excess of active NF-{kappa}B in severe uncontrolled asthma may impair the antiinflammatory action of GC and suggest the need to perform more mechanistic studies on the pathogenesis of severe asthma.


   Acknowledgments
 
R.G. has no declared conflict of interest; P.C. has no declared conflict of interest; M.M. has no declared conflict of interest; A.B. has no declared conflict of interest; G.C. has no declared conflict of interest; C.G. has no declared conflict of interest; I.V. has no declared conflict of interest; J.B. has no declared conflict of interest; G.B. has no declared conflict of interest; A.M.V. has no declared conflict of interest.

The authors thank P. Atger for reprographic services.


   FOOTNOTES
 
Supported by a grant from the Délégation à la Recherche Clinique de Montpellier and by a joint grant from CNR (Italy) and INSERM (France).

Received in original form May 28, 2002; accepted in final form July 30, 2003


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