INTRODUCTION

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Asthma is a chronic inflammatory disease of the airways, in which many inflammatory cells are involved and a large number of inflammatory genes are overexpressed (1). These genes encode (1) proinflammatory cytokines, including interleukin-1 (IL-1) and tumor necrosis factor- (TNF-), which amplify pulmonary inflammation; (2) chemokines, such as interleukin-8 (IL-8), macrophage inflammatory protein-1 (MIP-1), macrophage chemotactic protein-3 (MCP-3), RANTES (regulated on activation normal T-cell expressed and secreted) and eotaxin, which are chemotactic for leukocytes; (3) adhesion factors, including intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin, which play a cardinal role in leukocyte recruitment, margination, diapedesis, and transepithelial migration; and (4) inflammatory enzymes, such as cytosolic phospholipase A2 (cPLA2), inducible cyclooxygenase (COX-2), and inducible nitric oxide synthase (iNOS), which generate inflammatory mediators (2). All these inflammatory genes have been shown to contain B sites for the transcription factor, nuclear factor B (NF-B), within their promoter or enhancer and therefore to depend on NF-B for their expression, suggesting that this transcription factor could play a key role in the pathophysiology of asthma (2).

NF-B complexes are composed of five distinct DNA-binding subunits, called p50, p52, p65/RelA, c-Rel/Rel, and RelB (3). Although the most common NF-B complex is an heterodimer composed of the p65 and p50 subunits, the different family members can associate in various homo- or heterodimers through a highly conserved N-terminal sequence, called Rel homology domain (3). Inactive NF-B complexes are present in the cytosol associated with inhibitory proteins of the IB family, including IB-, IB-, IB-, p100, and p105 (3, 4). Following various stimuli, such as viruses, bacteria, prooxidants, and proinflammatory cytokines, IB proteins are first phosphorylated, and then rapidly degraded by the proteasome, allowing NF-B nuclear translocation and transcriptional initiation of NF-B-dependent genes (5).

Recently, increased NF-B activity (p65- and p50-containing complexes) has been demonstrated in macrophages of induced sputum and in bronchial epithelial cells of patients with stable asthma as compared with normal subjects, reinforcing the assumption that persistence of NF-B activity could be of particular importance in the pathogenesis of the chronic inflammation in asthma (6). Moreover, mice deficient in p50 are incapable of mounting eosinophilic airway inflammation when sensitized and challenged with ovalbumin (7). Although these recent studies allowed researchers to confirm the crucial role of NF-B in asthma, major issues remain unsolved. Is there a relation between the intensity of NF-B activity and the severity of the disease? Is there a relation between the kinetics of NF-B activity and the course of the disease? Are the NF-B complexes involved in asthma classic p65-p50 heterodimers? How is the sustained NF-B activity acquired in constitutive cells of the lung?

To solve these issues, it is necessary to use and repeat invasive procedures, such as bronchoalveolar lavages and bronchial brushings. Therefore, the use of an adequate model of asthma rather than asthmatic subjects appears to be appropriate. Heaves is a common, naturally occurring syndrome of horses, which is characterized by episodes of acute airway obstruction (crises) followed by periods of disease remission (8). Crisis develops within several hours of exposing heaves-susceptible horses to moldy hay, indicating involvement of an hypersensitivity response to inhaled antigens that grow in damp hay. Antigens commonly implicated are borne by spores of Aspergillus fumigatus, Faenia rectivirgula, and Thermoactinomyces vulgaris. Clinical remission occurs after antigens' eviction, when horses are pastured or stabled in a controlled environment. Heaves shares characteristic features with asthma, namely, recurrent airway obstruction, airway hyperresponsiveness, and chronic airway inflammation (8). Accordingly, heaves is considered to be the closest naturally occurring disease described in animals that parallels human asthma (9).

Here, bronchial cells (BCs) were obtained by bronchial brushing in healthy horses and in heaves-affected horses during and 21 d after the crisis. The relation between the level of NF-B activity in BCs and the degree of respiratory dysfunction was studied, and NF-B complexes were unambiguously characterized by the use of a modified protein extraction method.

	METHODS

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Experimental Animals

Twelve horses (521 ± 67 kg; 14.7 ± 2.4 yr; mean ± SD), with history and clinical signs of heaves were used. These horses were typically developing airway obstruction when housed in a barn and fed moldy hay, and entering in clinical remission once pastured or stabled in a controlled environment. One month before the experiment, they underwent a thorough clinical examination, including electrocardiogram, arterial blood gas analysis, hematology, endoscopy of the airways, tracheobronchial lavage, and pulmonary scintigraphy. This allowed the diagnosis to be made that they suffered from heaves and were free of any other health problem. Nine healthy horses (595 ± 43 kg; 8.5 ± 1.7 yr), i.e., without any airway obstruction when housed in barn and fed moldy hay, were used as control animals. Experimental horses did not receive any treatment during the month preceding the experiments.

Pulmonary Function Tests

Maximal change in pleural pressure (maxPpl) was measured using an esophageal balloon sealed over the end of a semi-rigid Teflon catheter (4 mm interior diameter, 6 mm outer diameter, 220 cm long), passed into the distal third of the esophagus and connected to a differential pressure transducer (Valydine DP45-18; Validyne Engineering, Northridge, CA). Respiratory flow was measured using a pneumotachograph (No. 4 Fleisch) connected to an airtight face mask and coupled to a differential pressure transducer (Valydine DP45-14). A computer provided with lung function software (Po Ne Mah System; Gould Instrument System, Valley View, OH), integrated the flow signal to determine tidal volume, and calculated dynamic compliance (C_dyn) and total pulmonary resistance (R_l) from the simultaneous measurements of pleural pressure changes and respiratory flow. More technical details are reported elsewhere (10).

Experimental Protocol

Bronchial cells of heaves-affected horses were obtained by bronchial brushing on two separate occasions: first, 24 h after the onset of a crisis, and then, 21 d after removal from the causative environment. To obtain a crisis, the horses were stabled and underwent a natural challenge with moldy hay. The horses were considered to be in crisis when their parameters of mechanics of breathing were within the following limits: maxPpl  2.00 kPa, RL  0.2 kPa · L¹ · s and C_dyn  8 L · kPa¹. Eviction of the antigenic agents was obtained by pasturing the horses, or by stabling them on dust-free bedding and feeding. Healthy horses were investigated twice with an interval of 21 d. The protocol was approved by the Ethics Committee of the Faculty of Veterinary Medicine of the University of Liège.

Bronchial Brushings

Horses were premedicated intravenously with romifidine 0.01 mg/kg (Sedivet; Boehringer Ingelheim, Ingelheim, Germany). Bronchoscopy was performed with a 9-mm-diameter bronchoscope (Pentax, Breda, The Netherlands) using a transnasal approach. The brushing was performed in 10 different places, from the main bronchi to the fourth generation airways, by inserting a cytology brush (Cook Veterinary Products, Eight Mile Plains, Australia) into the different segments. Bronchial cells were obtained using 20 gentle upward and downward strokes of the brush against the airways wall. Care was taken to avoid bleeding.

Cells Preparation and Immunostaining

After retracting the brush into its protective sheath and removing it from the bronchoscope channel, collected cells were dislodged by shaking the brush into 15-ml conical tubes containing ice-cold RPMI 1640 medium (Life Technologies, Merelbeke, Belgium) supplemented with 1% glutamine, 10% fetal bovine serum, gentamycin 50 µg/ml, and amphotericin B 10 µg/ml. The harvested cell suspension was filtered through gauze to remove mucus. The cells were then centrifuged at 800 g for 5 min, and the pellet was resuspended in Laboratory of Human Carcinogenesis (LHC-8) complete medium without hydrocortisone (Biofluids Inc., Rockville, MD), supplemented with amphotericin B 10 µg/ml. The cells were then incubated at 37° C in a 5% CO₂-95% air mixture for 3 h before protein extraction. Cell density was assessed by the use of a hemacytometer, and cell viability was evaluated by trypan blue exclusion. Fixed preparations of collected cells were obtained using a cytocentrifuge, and the epithelial nature of these cells was assessed by use of immunohistochemical labeling of cytokeratin-containing cells. Cytokeratin was detected with a monoclonal mouse anti-human cytokeratin antibody (Dako, Glostrup, Denmark). Afterwards, a three-step procedure using streptavidin-biotin-peroxidase- antiperoxidase detection system with 3-amino-9-ethylcarbazole as a chromogen for demonstration of peroxidase and hematoxylin as a counterstain was used (LSAB+; Dako). In detail, slides were first immersed for 10 min in a 3% solution of H₂O₂ in absolute ethanol to quench endogenous peroxidase activity. Nonspecific staining was reduced by immersion in 20% swine serum for 10 min, before application of the primary antibody (1/50) for 30 min at 37° C. This step was followed by successive incubations for 15 min with swine anti-mouse IgG and with peroxidase-streptavidin complex. Between each step, sections were thoroughly washed in Tris-buffered saline for 1 min (pH 7.4), then incubated for 5 min with chromogen, rinsed in tap water and counterstained with Mayer's hematoxylin stain. Negative controls omitting the incubation with the primary antibody, were done for each specimen.

Cytoplasmic and Nuclear Protein Extraction

Cytoplasmic and nuclear protein extracts were prepared as previously described (11). Cytoplasmic buffer contained 10 mM Hepes, pH 7.9, 10 mM KCl, 2 mM MgCl₂, 0.1 mM ethylenediaminetetraacetic acid (EDTA), 0.2% (vol/vol) Nonidet P-40, and 1.6 mg/ml protease inhibitors (Complete; Boehringer Mannheim, Mannheim, Germany). The pelleted nuclei were resuspended in 20 mM Hepes, pH 7.9, 1.5 mM MgCl₂, 0.2 mM EDTA, 0.63 M NaCl, 25% (vol/vol) glycerol, and 1.6 mg/ml protease inhibitors (nuclear buffer), incubated for 20 min at 4° C, and centrifuged for 30 min at 14,000 rpm (Centrifuge 5415C; Eppendorf, Hamburg, Germany). Protein amounts were quantified with the Micro BCA Protein Assay Reagent Kit (Pierce, Rockford, IL). In some experiments, cytoplasmic and nuclear protein extraction was modified, according to the method described by Franzoso and coworkers (12), by supplementing buffers with 3 mM of the protease inhibitor diisopropyl fluorophosphate (DFP; Sigma Chemical Co., Bornem, Belgium).

Electrophoretic Mobility Shift Assays (EMSA)

Binding reactions were performed for 30 min at room temperature with 5 µg of nuclear proteins in 20 mM Hepes, pH 7.9, 10 mM KCl, 0.2 mM EDTA, 20 % (vol/vol) glycerol, 1% (wt/vol) acetylated bovine serum albumin, 3 µg of poly(dI-dC) (Amersham Pharmacia Biotechnology, Little Chalfont, UK), 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, and 100,000 cpm of ³²P-labeled double-stranded oligonucleotide probes. Probes were prepared by annealing the appropriate single-stranded oligonucleotides (Eurogentech, Liège, Belgium) at 65° C for 10 min in 10 mM Tris, 1 mM EDTA, 10 mM NaCl, followed by slow cooling to room temperature. The probes were then labeled by end-filling with the Klenow fragment of Escherichia coli DNA polymerase I (Boehringer Mannheim, Mannheim, Germany), with phosphorus-32-deoxyadenosine triphosphate (³²P-dATP) and phosphorus-32-deoxycytidine triphosphate (³²P-dCTP) (Dupont-New England Nuclear [NEN] Life Science Products, Les Ulis, France). Labeled probes were purified by spin chromatography on G-25 columns. DNA-protein complexes were separated from unbound probe on 4% native polyacrylamide gels at 150 V in 0.25 M Tris, 0.25 M sodium borate, and 0.5 mM EDTA, pH 8.0. Gels were vacuum-dried and exposed to Fuji X-ray film at 80° C for 12 h. The amount of specific complexes was determined by photodensitometry of the autoradiography. To confirm specificity, competition assays were performed with a 50-fold excess of unlabeled wild-type probes and with mutated probes. For supershifting experiments, 1.5 µl of each antibody was incubated with the extracts for 30 min before addition of the radiolabeled probe. The sequences of the oligonucleotides used in this work were as follows: wild-type palindromic B probe (13), 5'-TTGGCAACGGCAGGGGAATTCCCCTCTCCTTAGGTT-3'; mutated palindromic B probe, 5'-TTGGCAACGGCAGATCTATTCCCCTCTCCTTAGGTT-3'.

Immunoblots

Nuclear protein extracts (10 µg) were added to a loading buffer (10 mM Tris-HCl, pH 6.8, 1% [wt/vol] sodium dodecyl sulfate [SDS], 25% [vol/vol] glycerol, 0.1 mM -mercaptoethanol, 0.03% [wt/vol] bromophenol blue), boiled, and run on a 10% sodium dodecyl sulfate/ polyacrylamide gel electrophoresis (SDS/PAGE) gel. After electrotransfer to polyvinylidene difluoride membranes (Boehringer Mannheim) and blocking overnight at 4° C with 20 mM Tris, pH 7.5, 500 mM NaCl, 0.2 (vol/vol) Tween 20 (Tris/HCl/Tween) plus 5 % (wt/vol) dry milk, the membranes were incubated for 1 h with the first antibody (1:1,000 dilution), washed, then incubated with peroxidase-conjugated goat anti-rabbit IgG (1:5,000 dilution) (Kirkegaard & Perry Laboratories Inc., Gaithersburg, MD) for 45 min. The reaction was revealed with the enhanced chemiluminescence detection method (ECL kit; Amersham Pharmacia Biotechnology).

Anti-NF-B and Anti-ICAM-1 Antibodies

The anti-NF-B antibodies used were: (1) a rabbit antibody recognizing an NH₂-terminal peptide (amino acids 1-12) of p50 (Upstate Biotechnology, Inc., Lake Placid, NY); (2) a rabbit antibody directed to the 13 COOH-terminal amino acids of p65 (Upstate Biotechnology, Inc.); (3) a rabbit antibody directed to a COOH-terminal peptide (amino acids 531-550) of p65 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA); (4) a rabbit p65 NH₂-terminal peptide antibody (Santa Cruz Biotechnology, Inc.); (5) a mouse monoclonal antibody directed against amino acids 1-444 of human NF-B p52 subunit (Upstate Biotechnology, Inc.); (6) a rabbit antibody recognizing a NH₂-terminal peptide of Rel (Santa Cruz Biotechnology, Inc.); (7) a rabbit antibody directed against a COOH-terminal peptide of RelB (Santa Cruz Biotechnology, Inc.). The anti-ICAM-1 antibody used was a rabbit antibody recognizing the epitope corresponding to amino acids 258-365 mapping within the extracellular domain of ICAM-1 of human origin (Santa Cruz Biotechnology, Inc.).

Statistical Analysis

To study the correlation between NF-B activity in BCs, as assessed by photodensitometry, and maxPpl, C_dyn and RL, standard least-square linear regressions were carried out. Coefficients of correlation (r) were presented as measures of linear association for regression relationships. Significant differences of the slopes from zero were determined using a two-tailed Student's t test. A p value of less than 0.05 was considered as significant. The differences in cell viability in healthy and heaves-affected horses were assessed by the use of a Student's t test for unpaired data.

	RESULTS

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Cell Number, Viability, and Type

The number of harvested cells averaged 15.2 ± 8.3 (mean ± SD) million cells/animal (range, 6 to 26 million). The viability of harvested cells averaged 22.2 ± 10.3%, 18.7 ± 9.6%, and 24.4 ± 8.4%, in healthy horses, heaves-affected horses in crisis, and 21 d after eviction from the causative environment, respectively (Table 1). Theses percentages were not significantly different. The percentage of cells identified as bronchial epithelial cells was always > 95. The differential cells counts of the bronchial brushing samples obtained from healthy and heaves-affected horses are given in Table 1.

Relation between NF-B Activity and Lung Function

Nuclear extracts prepared from BCs of healthy horses demonstrated a weak basal NF-B-binding activity (Figure 1, lane 5; Figure 2A, lanes 1-4). NF-B activity was much greater in extracts from cells obtained in heaves-affected horses during crisis (Figure 1, lane 6; Figure 2A, lanes 5, 7, and 9). Active NF-B complexes observed in BCs of healthy and heaves-affected horses did not comigrate (Figure 1, lanes 5 and 6 ). Cells of heaves-affected horses yielded large and diffuse NF-B complexes, which migrated faster than those observed in cells of healthy horses. DNA-binding competition experiments using 50-fold excess of unlabeled wild-type and mutated palindromic  probes confirmed specificity of NF-B binding in cells of both healthy and heaves-affected horses (Figure 1, lanes 1-4). Twenty-one days after eviction of the causative agents, NF-B activity returned to basal level in three heaves-affected horses, was maintained at high levels in six animals, and reached an intermediate level in three individuals. Examples are given in Figure 2A (lanes 6, 8, and 10). When NF-B activity was maintained at intermediate or high levels, diffuse and faster migrating complexes were observed (Figure 2A, lanes 8 and 10). When NF-B activity returned to basal level, NF-B complexes comigrated with those observed in BCs of healthy horses (Figure 2A, lane 6).

View larger version (51K): [in this window] [in a new window]   Figure 1.   NF-B activity in bronchial brushing samples obtained from healthy and heaves-affected horses. Electrophoretic mobility shift assays were performed with nuclear protein extracts prepared from bronchial cells of one healthy horse (lane 5) and one heaves-affected horse during crisis (lane 6 ). The open and solid arrows indicate specific NF-B complexes revealed in cells of the healthy horse and the heaves-affected horse, respectively. Specificity of NF- complexes was confirmed in both healthy horse (lanes 1 and 2) and heaves-affected horse (lanes 3 and 4) by competition experiments using the same nuclear extracts incubated with a 50-fold excess of unlabeled wild-type palindromic B probes (lanes 1 and 3) and mutated palindromic B probes (lanes 2 and 4). This experiment was representative of 12 similar experiments.

View larger version (59K): [in this window] [in a new window]   Figure 2.   Relation between specific NF-B activity displayed by bronchial cells obtained by brushing and lung function in healthy and heaves-affected horses. (A) NF-B DNA-binding activity of nuclear protein extracts obtained from bronchial cells of four healthy horses (lanes 1 to 4) and three heaves-affected horses (lanes 5 to 10). Each heaves-affected horse was investigated on two occasions: during crisis (lanes 5, 7, and 9) and 21 d after removal from the causative environment (lanes 6, 8, and 10). The open arrow indicates specific NF-B complexes revealed in cells of all healthy horses and of one heaves-affected horse which completely recovered 21 d after crisis. The solid arrow indicates specific faster migrating NF-B complexes revealed in cells of heaves-affected horses, during crisis and 21 d after if they did not completely recover. Histograms show the amounts of specific NF-B complexes as determined by phosphodensitometry of the autoradiography. (B) Maximal change in pleural pressure (maxPpl, kPa), dynamic compliance (Cdyn, L · kPa1), and total pulmonary resistance (RL, kPa · L1 · s) measured in the seven investigated horses 1 h before each bronchial brushing. (C ) Relationships between values of maxPpl, Cdyn, and RL, and intensity of corresponding NF-B bands (r = correlation coefficient). This experiment is representative of three similar experiments performed in 12 heaves-affected horses and nine healthy horses.

In healthy horses, maxPpl, C_dyn, and RL averaged 0.88 kPa, 14.9 L · kPa¹, and 0.05 kPa · L¹ · s, respectively. In heaves-affected horses during crisis, these parameters averaged 4.22 kPa, 6.5 L · kPa¹, and 0.32 kPa · L¹ · s, respectively. Three weeks after the crisis, horses whose cells yielded high levels of NF-B activity had average values of maxPpl, C_dyn and RL similar to those observed during the crisis (4.17 kPa, 5.9 L · kPa¹, and 0.29 kPa · L¹ · s, respectively), while horses showing basal NF-B activity had pulmonary function values similar to those observed in healthy horses (1.17 kPa, 13.5 L · kPa¹, and 0.07 kPa · L¹ · s, respectively). Horses with intermediate NF-B activity had also intermediate levels of maxPpl, C_dyn and RL (2.72 kPa, 9.5 L · kPa¹, and 0.16 kPa · L¹ · s, respectively). Examples are shown in Figure 2B.

To confirm the apparent link between lung function and NF-B-binding activity, correlations between values of maxPpl, C_dyn and RL, and intensity of corresponding NF-B bands, as measured by photodensitometry, were calculated. These regression analyses were carried out with the results obtained from three separate gels. Correlation coefficients between (a) maxPpl (b) C_dyn, and (c) RL, and intensity of NF-B bands, were (a) 0.85 (first gel), 0.79 (second gel), and 0.88 (third gel); (b) 0.88, 0.76, and 0.85; and (c) 0.76, 0.84, and 0.84, respectively. Results obtained from a representative gel are shown in Figure 2C.

Characterization of NF-B Complexes

Supershifting experiments performed with antibodies directed against the various members of the NF-B family (i.e., p50, p65, p52, Rel, and RelB) showed that the retarded complex observed in nuclear protein extracts from BCs of healthy horses, and heaves-affected horses in complete functional remission, contained p65 and p50 subunits (Figure 3). Weak Rel supershifts were occasionally observed (data not shown).

View larger version (48K): [in this window] [in a new window]   Figure 3.   Characterization of specific active NF-B complexes yielded by bronchial cells of healthy horses and heaves-affected horses in complete remission. Supershift analysis was conducted with antibodies directed against p50 (lane 2), a COOH-terminal peptide of p65 (amino acids 531-550) (lane 3), a NH2-terminal peptide of p65 (lane 4), p52 (lane 5), Rel (lane 6 ), and RelB (lane 7). The specific complexes are indicated by an open arrow. Supershifts of p50 and p65 are indicated by open arrowheads. This experiment is representative of six similar experiments.

Nuclear extracts prepared from BCs of heaves-affected horses, during crisis and 21 d later, except when horses completely recovered, demonstrated large, diffuse, and faster migrating specific NF-B complexes (Figure 1, lane 6; Figure 2A, lanes 5 and 7-10). To characterize these complexes, supershift assays were performed (Figure 4A). Anti-p50 antibodies, anti-p65 antibodies directed against the NH₂-terminal peptide, and anti-Rel antibodies weakly supershifted the faster migrating NF-B complexes. However, Rel supershifts were rarely observed. Anti-p52 antibodies, anti-RelB antibodies, and anti-p65 antibodies directed against the COOH-terminal peptide did not supershift the faster migrating NF-B complexes. This suggested that these particular complexes contained a form of p65 which was truncated at the COOH-terminus and that the truncation occurred after the Rel-homology domain, preserving the ability of this protein to dimerize and bind DNA.

View larger version (31K): [in this window] [in a new window]   Figure 4.   Characterization of specific active NF-B complexes yielded by bronchial cells of heaves-affected horses, during crisis and 21 d later when horses did not completely recover. (A) Supershift analysis was conducted with antibodies directed against p50 (lane 2), the 13 COOH-terminal amino acids of p65 (lane 3), a COOH-terminal peptide of p65 (amino acids 531-550) (lane 4), a NH2-terminal peptide of p65 (lane 5), p52 (lane 6 ), Rel (lane 7), and RelB (lane 8). The specific complexes are indicated by a solid arrow. Supershifts of p50, p65, and Rel are indicated by solid arrowheads. (B) To better characterize NF-B complexes, nuclear protein extraction was modified by supplementing buffers with 3 mM of DFP, a potent protease inhibitor, and electrophoretic mobility shift and supershift assays were performed on harvested extracts. This procedure allowed the conversion of the diffuse and faster migrating NF-B complexes as seen in lane 1 into more slowly migrating complexes as seen in lane 2. Supershift analysis was conducted to characterize converted complexes, with antibodies directed against p50 (lane 3), the 13 COOH-terminal amino acids of p65 (lane 4), a COOH-terminal peptide of p65 (amino acids 531-550) (lane 5), a NH2-terminal peptide of p65 (lane 6 ), p52 (lane 6 ), Rel (lane 8), and RelB (lane 9). The specific complexes are indicated by solid arrows. Supershifts of p50, p65, and Rel are indicated by solid arrowheads. This experiment was representative of three similar experiments.

To ascertain this hypothesis, cytoplasmic and nuclear protein extraction was modified by supplementing buffers with 3 mM of DFP, a potent protease inhibitor, and electrophoretic mobility shift and supershift assays were performed on harvested extracts (Figure 4B). This procedure allowed the conversion of the diffuse and faster migrating NF-B complexes into complexes that comigrated with those observed in cells from healthy horses. Converted NF-B complexes were made of two bands: an intense upper band and a fainter lower band. Supershift assays demonstrated that the upper band, which was particularly displaced by anti-p65 antibodies (antibodies directed against COOH- and NH₂-terminal peptides), but not by p50 antibodies, was formed mainly of p65-p65 homodimers whereas the lower band, which reacted only with anti-p50 antibodies, corresponded to p50-p50 homodimers. Supershifts with anti-Rel antibodies were rarely observed, and anti-p52 and anti-RelB antibodies did not supershift the converted NF-B complexes. To ascertain that the inability of the anti-p50 antibodies to supershift the upper specific band was not due to a weak interaction of the antibodies with the prototypical p50-p65 heterodimers, supershift experiments using these anti-p50 antibodies were performed with nuclear extracts prepared from stimulated ovarian carcinoma cells (OVCAR), which are known to contain high amounts of p50-p65 heterodimers, and low amounts of p50 homodimers (14). In these nuclear extracts, all NF-B complexes were completely displaced by anti-p50 antibodies (data not shown). Thus, these data indicate that nuclear extracts prepared from BCs of heaves- affected horses contain authentic p65 homodimers.

Presence of p50 and p65 proteins in nuclear extracts prepared from BCs of healthy and heaves-affected horses, during crisis and 21 d later was confirmed by immunoblots (Figure 5). Only small amounts of p50 and p65 were revealed by immunoblot in nuclear extracts obtained from cells of healthy horses (lanes 1 and 4 ). On the contrary, important amounts of these proteins were observed in extracts from cells of heaves-affected horses, either during crisis (lanes 2 and 5) or 21 d after removal from the causative environment (lanes 3 and 6), except when horses completely recovered.

View larger version (33K): [in this window] [in a new window]   Figure 5.   Representative p50 and p65 immunoblots performed with nuclear extracts from bronchial cells of healthy horses (lanes 1 and 4), and heaves-affected horses, during crisis (lanes 2 and 5) and 21 d after removal from the causative environment (lanes 3 and 6 ). Arrows indicate the position of the specific bands. This experiment is representative of three similar experiments.

ICAM-1 Expression

Presence of ICAM-1 in cytoplasmic extracts prepared from BCs of healthy and heaves-affected horses, during crisis and 21 d later, was assessed by immunoblots (Figure 6). Only small amounts of ICAM-1 were revealed by immunoblot in cytoplasmic extracts obtained from cells of healthy horses (lane 1). On the contrary, important amounts of these proteins were observed in extracts from cells of heaves-affected horses during crisis (lane 2). Twenty-one days after removal from the causative environment, ICAM-1 expression reached intermediate levels (lane 3).

View larger version (29K): [in this window] [in a new window]   Figure 6.   Representative ICAM-1 immunoblot performed with cytoplasmic extracts from bronchial cells of healthy horses (lanes 1), and heaves-affected horses, during crisis (lanes 2) and 21 d after removal from the causative environment (lanes 3). Arrows indicate the position of the specific bands. This experiment is representative of three similar experiments.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

NF-B, which is thought to play a cardinal role in transcriptional initiation of the inflammatory genes, is overexpressed and more active in the bronchial epithelium and macrophages of patients with stable asthma compared with healthy subjects (6). In the present report, we confirm these findings in an animal model of asthma and demonstrate that the level of NF-B activity in BCs is highly correlated to the degree of lung dysfunction, and that the kinetics of NF-B activity is strongly related to the course of the disease. These findings suggest that the level of NF-B activity could determine the severity of respiratory dysfunction in asthma and asthmalike diseases and confirm the relevance of NF-B as a potential target in asthma therapy.

Bronchial cells of healthy horses and heaves-affected horses in complete functional remission demonstrated a weak basal NF-B activity, involving p50- and p65-containing complexes. These results are in accordance with those previously reported by Hart and coworkers (6) in asthmatic patients. Only few cell types display a constitutive NF-B activation, namely, mature B-cells (15), thymocytes (16), some monocytic cell lines (17), and certain neurons (18). Therefore, it would be hazardous to state that BCs display a constitutive NF- activity, as well. Because BCs are in permanent contact with inflammatory stimuli, such as inhaled oxidants, it is more likely that NF-B activity is perpetually induced in a small proportion of these cells rather than constitutive.

Nuclear extracts prepared from BCs of heaves-affected horses, during crisis and 21 d later, except when horses completely recovered, demonstrated diffuse and faster migrating NF-B complexes, characterized by truncation of the COOH-terminal part of p65 subunits. Protein extraction in the same cells in the presence of DFP, a particular protease inhibitor, allowed the detection of unmodified NF-B complexes which comigrated with those observed in BCs of healthy horses, indicating that truncation of p65 subunits occurs during protein extraction rather than in living cells. DFP specifically inhibits a family of serine proteases (i.e., the serprocidins), including cathepsin G, elastase, and proteinase-3 (19), which are exclusively contained within azurophil granules of monocytes, neutrophils, and certain promonocytic and promyelocytic cells (12, 22). Accordingly, one may assume that truncation of p65 subunits in BCs of heaves-affected horses was due to the release, during protein extraction, of active streptocidins from lysosomes of monocytic cells and granulocytes, especially neutrophils, which were more present in cell samples from heaves-affected horses when compared with those from healthy horses. Increased NF-B activity has been previously reported in induced sputum of asthmatic patients (6). NF-B complexes shown in this prior study were undoubtedly diffuse, suggesting that faster migrating NF-B complexes, rather than unmodified ones, may have been present in induced sputum. These observations taken together lead us to recommend the use of DFP when preparing cytoplasmic and nuclear protein extracts from induced sputum, bronchoalveolar lavage, and bronchial brushings samples, which all may contain neutrophils and cells of the monocytic lineage.

Protein extraction of BCs in the presence of DFP also allowed the characterization of the faster migrating NF-B complexes observed in heaves-affected horses. Unexpectedly, these complexes were mainly p65 homodimers and, to a lesser extent, p50 homodimers. Although the presence of endogenous p65 homodimers has been established in certain cells, such as T cells and endothelial cells (26, 27), little is known about the physiological significance of these uncommon complexes. Transcription of some genes, such as ICAM-1 and IL-8, critically depends on an atypical B site which preferentially binds p65 homodimers, suggesting that the p65 homodimer is the key transactivator of these genes (27, 28). These observations suggest that, in BCs, p65 homodimers could induce transcription through B sites that are not appreciably recognized by the classic p65-p50 heterodimers, and therefore regulate the expression of a subset of genes characteristic of asthma and asthmalike diseases. This is further supported by the findings (1) that expression of ICAM-1 is greatly increased in bronchial epithelial cells of asthmatic patients when compared with patients with chronic bronchitis, another inflammatory disease of the airways (29), and (2) that ICAM-1 expression paralleled p65 homodimers activity in BCs of healthy and heaves-affected horses. Moreover, p50^/ mice, which lack p50 homodimers and p65-p50 heterodimers, continue to overexpress ICAM-1 when sensitized and challenged with ovalbumin, demonstrating that ICAM-1 expression in asthmalike diseases is not dependent on the classic p65-p50 heterodimers (7).

Twenty-one days after removal of the antigen, NF-B activity was maintained at high or moderate levels in BCs of the majority of heaves-affected horses, indicating that NF-B activity in asthmalike diseases does not necessarily require the continuous presence of the etiologic agent. An intriguing question is how this persistent NF-B activity in BCs is acquired. Although it is likely that NF-B activity in BCs lasts as long as inflammatory cells or long-acting inflammatory mediators are present in the airways, other hypotheses cannot be ruled out. These hypotheses mainly appear in the light of studies devoted to mature B cells. Mature B cells exhibit constitutive NF-B activity (30). In these cells, classic p50-p65 heterodimers are replaced by typical p50-Rel and p52-RelB heterodimers, which are maintained in the nucleus in an active form (34, 35). Constitutive NF-B activity in mature B cells has been explained by the facts that inhibition of p50-Rel and p52-RelB dimers by IB- is less efficient than that of p50-p65 dimers (3, 36) and that the half-life of IB-, and therefore the ability of this inhibitor to sequester NF-B complexes in the cytosol, is significantly decreased in mature B cells (3, 35). Active Rel-containing NF-B complexes have only been occasionally observed in the present study, suggesting that this subunit is not involved in the persistence of NF-B activity in BCs of heaves-affected horses. On the other hand, previous studies suggest that p65 homodimers, like p50-Rel and p52-RelB heterodimers, have a low affinity for IB- (26), indicating that the presence of activated p65 homodimers in the nucleus of BCs of heaves-affected horses could account for the sustained NF-B activity in these cells. Alternatively, a continuous signaling event could occur in BCs of heaves-affected horses, leading to increased degradation of IB-, or other proteins of the IB family, and to subsequent long-term activation of NF-B. This continuous signaling could be due either to acquired defects in the intracellular pathway that regulates IB- degradation, or to an autocrine stimulation by proinflammatory cytokines, such as IL-1 and TNF-, whose expression mainly depends on NF-B activity.