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Published ahead of print on April 15, 2004, doi:10.1164/rccm.200305-659OC
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American Journal of Respiratory and Critical Care Medicine Vol 170. pp. 141-147, (2004)
© 2004 American Thoracic Society

Original Article

Histone Acetylase and Deacetylase Activity in Alveolar Macrophages and Blood Mononocytes in Asthma

Borja G. Cosío, Buphinder Mann, Kazuhiro Ito, Elen Jazrawi, Peter J. Barnes, K. Fan Chung and Ian M. Adcock

Department of Thoracic Medicine, National Heart and Lung Institute, Imperial College Faculty of Medicine, London, United Kingdom

Correspondence and requests for reprints should be addressed to Ian Adcock, Ph.D., Thoracic Medicine, National Heart and Lung Institute, Dovehouse Street, London SW3 6LY, UK. E-mail: ian.adcock{at}imperial.ac.uk


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Histone acetylation status is a key factor in the regulation of inflammatory gene transcription. We investigated the activity of histone acetylases (HAT) and deacetylases (HDAC), and the effect of glucocorticoids in alveolar macrophages (AM) and peripheral blood mononuclear cells (PBMC) from subjects with asthma. Bronchoalveolar lavage was performed in 10 patients with intermittent asthma, 8 with persistent asthma, and 10 healthy control subjects. PBMCs and granulocytes were isolated from six patients with mild and severe asthma, before and after a 7-day course of prednisolone (30 mg/day). AMs were isolated for HDAC assay or incubated with dexamethasone (1 µM). HAT activity was increased (1.43 ± 0.1 vs. 1.01 ± 0.1 standard units/10 µg, p < 0.05), and HDAC activity was reduced (3,031 ± 243 vs. 5,004 ± 164 arbitrary fluorescence units/10 µg, p < 0.001) in AMs of subjects with asthma compared with control subjects. Dexamethasone suppressed LPS-induced granulocyte macrophage-colony stimulating factor, tumor necrosis factor-{alpha}, and interleukin-8 release by 83 ± 1%, 51 ± 7% and 20 ± 9% (p < 0.001), respectively. Similar effects were seen on nuclear factor-{kappa}B inhibition, and interleukin-8 release was further reduced by the HDAC enhancer, theophylline (37 ± 6%). Prednisolone increased HDAC activity in PBMCs from subjects with mild asthma. The increased inflammatory response in asthma may be due to reduced HDAC and enhanced HAT activity. Glucocorticoids and theophylline may downregulate the inflammatory response by modulating HAT and HDAC activity, and nuclear factor-{kappa}B activation.

Key Words: inflammation • chromatin remodeling • theophylline • glucocorticoids • asthma

Asthma is an inflammatory disease of the airways which involves several inflammatory cells (T lymphocytes and eosinophils) and multiple inflammatory mediators (1). Macrophages and monocytes also play an important role in the regulation of the immune and inflammatory responses, although their exact role in asthma is unclear. Alveolar macrophages (AM) and peripheral blood mononuclear cells (PBMC) from subjects with asthma have been reported to display an activated phenotype, with upregulated surface expression of HLA-DR, CD35, and CD23 receptors (2, 3). However, Viksman and colleagues (4) reported evidence of activation of AMs, but not PBMs, in subjects with allergic rhinitis and asthma.

Histone acetyl-transferases (HAT) and deacetylases (HDAC) are families of enzymes that regulate chromatin structure, and through this, affect inflammatory gene expression (5). Acetylation of histones by coactivator proteins, such as CREB-binding protein (CBP), p300, and TAFII250, which possess intrinsic HAT activity, leads to unwinding of the DNA to allow transcription factors and RNA polymerase II to switch on gene transcription. Conversely, deacetylation of core histones is associated with transcriptional repression (6). Glucocorticoids are very effective in repressing inflammation in asthma, and glucocorticoid suppression of inflammatory genes requires, at least in part, recruitment of HDACs to the transcriptional activation complex by the glucocorticoid receptor (7). We have also previously shown that glucocorticoids can modulate antiinflammatory and proinflammatory cytokines in AMs and PBMs in patients with asthma (8).

We wished to explore the hypothesis that inflammation can be modulated in early stages of asthma through the alteration of the activity or expression of these enzymes. Ito and colleagues (9) have previously shown increased HAT activity and reduced HDAC activity in bronchial biopsies from subjects with asthma, which was reversed to normal in subjects taking inhaled steroids. An attempt to readdress this imbalance could be through drugs that increase HDAC activity or decrease HAT activity. We have shown that theophylline is a potent inductor of HDAC activity in cell lines and alveolar macrophages from subjects with asthma (10).

The objective of this study was, therefore, to investigate HAT and HDAC activity in AMs from subjects with asthma, and the effect of glucocorticoids administered in vivo and ex vivo on HDAC activity and cytokine release in these cells. We also studied the HDAC activity of PBMCs and granulocytes from patients with asthma having different degrees of severity, and the effects of systemic glucocorticoids on HDAC expression and activity. The results of this study have been partly reported in the form of an abstract (11).


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Subjects and Protocols
Comparison of HAT and HDAC activity in AMs from patients with asthma.
Eighteen patients with asthma were recruited for bronchoscopy. Asthma severity was defined according to the Global Initiative for Asthma criteria (12). Ten patients with asthma had intermittent symptoms of asthma requiring only treatment with inhaled ß-adrenergic agonist, albuterol, for relief of wheeze. The other eight patients with asthma had moderate persistent asthma, and were receiving regular inhaled glucocorticoids (500 µg/day of fluticasone or equivalent) for control of asthma. All patients demonstrated a greater than 15% improvement in FEV1 after 200 µg of albuterol, and airway responsiveness to a provocative concentration of methacholine producing a 20% fall in FEV1 (PC20) of less than 4 mg/ml. All patients were atopic, as defined by two or more positive skin prick tests to common allergens. Current smokers or exsmokers of more than 5 pack-years were excluded. Ten healthy nonatopic, nonsmoking individuals were also recruited as control subjects for bronchoscopy (Table 1) .


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  		TABLE 1. Characteristics of subjects undergoing bronchoscopy

 
Effect of glucocorticoids on HDAC activity.
In a separate experiment, six patients with mild persistent asthma, and six patients with severe persistent asthma receiving inhaled glucocorticoid (> 1,000 µg/day of fluticasone or equivalent), long-acting inhaled ß2 agonist, and oral glucocorticoids, if required (n = 3), for control of asthma, were studied. Venous blood was obtained from subjects before and after receiving a week-long course of 30 mg/day of oral prednisolone (subjects with mild asthma), or an additional 30 mg/day of prednisolone (subjects with severe asthma already on maintenance oral prednisolone). Prednisolone blood levels and cortisol were measured to ensure compliance to treatment. Exhaled nitric oxide (eNO) was also measured before and after treatment following a standardized method (13). A blood sample was also obtained from six healthy subjects as a control for PBMC baseline HDAC activity (Table 2) . The protocols were approved by the Royal Brompton Hospital and the National Heart and Lung Institute Ethics Committee, and all patients and volunteers gave written informed consent.


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  		TABLE 2. Characteristics of subjects in blood study

 
Fiberoptic Bronchoscopy, Bronchoalveolar Lavage Collection, and Macrophage Processing
This was performed essentially as previously described (14). Subjects attended the bronchoscopy suite at 8:30 A.M. after having fasted from midnight. Midazolam (2.5–5 mg intravenously) and alfentanyl (0.5 mg intravenously) were used for sedation during the procedure. Local anesthesia (2% lidocaine) was used on upper airways and larynx, and a fiberoptic bronchoscope (Olympus BF30; Key-Med, Southall, UK) was passed through the nasal passages into the trachea. Warmed 0.9% NaCl solution (60 ml, 4 times) was instilled into the right middle lobe of the lung to enable recovery of bronchoalevolar lavage (BAL) cells by gentle suction. BAL cells were centrifuged (500 x g for 10 minutes) and washed twice with Hanks' balanced salt solution (HBSS). Cytospins were prepared and stained with May-Grunwald stain for differential cell count. Cell viability was assessed using the trypan-blue exclusion method. BAL macrophages were either lysed as previously described (10), or isolated by plastic adhesion and the cells (3 x 105) incubated in 24-well plates in the presence of theophylline (10 µM), dexamethasone (1 µM), and/or Salmonella enteritidis LPS (10 µg/ml; Sigma, Poole, UK). Some of these experiments were also performed in the presence of IKK2 inhibitor (AS602868; Serono, Geneva, Switzerland), which has been previously shown to inhibit the nuclear factor-{kappa}B (NF-{kappa}B) pathway in cell lines (15, 16).

Isolation of Human PBMCs
Venous blood (60 ml) was collected from patients with asthma and healthy control subjects and diluted 1:1 with sterile HBSS. PBMCs were prepared from the whole blood by density centrifugation, as previously described (17). Twenty-five milliliters of diluted blood were layered on top of 12.5 ml of Ficoll-Hypaque-Plus (density, 1.077 g/ml; Amersham Biosciences, Amersham, UK) and centrifuged for 30 minutes at 1,100 x g at room temperature. The interface was collected and washed twice with phosphate-buffered saline (PBS). Cells were pelleted by centrifugation at 250 x g for 10 minutes and stained with Kimura dye for determination of total cell number. Cell viability, as determined by trypan blue exclusion, was uniformly greater than or equal to 97%. To obtain granulocytes, all layers in the density sedimentation above the red cell layer were removed, 10 ml of PBS and 3 ml of 3% Dextran T-500 were added to the layers, and the mixture was left at room temperature for 40 minutes. The supernatant was carefully removed and centrifuged at 200 x g for 10 minutes. The red cells were lysed by the addition of 2 ml of 10% HBSS in distilled H2O for 20 seconds, additional HBSS (minimum 12 ml) was added, and the contents of the tube were mixed and centrifuged at 1,800 rpm for 5 minutes. The cells were washed twice in HBSS (Ca2+- and Mg2+-free) containing 15% fetal calf serum (FCS). Kimura-stained cells were counted on a hemocytometer, resuspended at 5 x 106/ml, and viability checked using trypan blue.

Whole Blood Culture
Venous blood was collected in heparinized tubes and diluted 1:10 with RPMI 1640 (Gibco, Invitrogen, UK) containing 10% FCS, 100 U/ml penicillin, 100 µg/ml streptomycin, and 2 mM L-glutamine. The diluted blood was distributed in 1-ml aliquots into 24-well plates and incubated at 37°C with 5% CO2 for 24 hours. The plates were centrifuged at 1,800 rpm for 5 minutes, and the supernatants were removed and frozen at –70°C until analyzed.

Assay for Interleukin-8, Tumor Necrosis Factor-{alpha}, and Granulocyte Macrophage-Colony Stimulating Factor
Interleukin-8 (IL-8), granulocyte macrophage-colony stimulating factor (GM-CSF), and tumor necrosis factor-{alpha} (TNF-{alpha}) expression were measured by sandwich ELISA according to manufacturer's recommendations (R&D Systems Europe, Abingdon, UK).

Western Blotting
Nuclear extracts were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting using enhanced chemiluminescence (ECL; Amersham Biosciences). Proteins were size-fractionated by SDS-PAGE and transferred to Hybond-ECL membranes. Inmunoreactive bands were detected by ECL using specific antibodies obtained from Santa Cruz Biotechnology (Santa Cruz, CA), as previously described (7).

HDAC Activity
HDAC activity of nuclear extracts was measured with a nonisotopic assay using a fluorescent derivative of epsilon-acetyl lysine (HDAC Fluorescent Activity Assay Kit; BIOMOL, Plymouth Meeting, PA). This assay is based on the Fluor de Lys (Fluorogenic Histone Deacetylase Lysyl Substrate/Developer) Substrate and Developer combination. The assay was performed exactly as recommended by the manufacturer, and emitted light detected at 460 nm in a fluorometric plate reader.

HAT Activity
HAT activity of nuclear lysates was determined with an indirect ELISA for the detection of acetyl residues, following manufacturer recommendations (HAT Assay Kit; Upstate Biotechnology, Charlottesville, VA).

Statistics
Results are expressed as the mean ± SEM. Changes in macrophage secretory products from subjects with asthma and control subjects were compared using one-way analysis of variance (ANOVA). Comparisons between experimental groups were performed using the Mann-Whitney U test, and the paired t test was used to examine the effects of prednisolone. All statistical testing was performed using a two-sided 5% level of significance using GraphPad Prism software (GraphPad Software Inc., San Diego, CA).


   RESULTS
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 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Patient Characterization
Baseline characteristics and investigation results of the subjects studied are summarized in Table 1. The number of macrophages/ml of recovered BAL fluid was significantly higher in subjects with moderate asthma receiving inhaled corticosteroids (ICS).

Characteristics of the subpopulation of subjects in whom blood studies were performed are summarized in Table 2. Three of the subjects in the group with severe asthma were receiving maintenance prednisolone (5, 10, and 15 mg/day). There was a trend toward an improvement in FEV1 (from 1.29 ± 0.3 to 1.74 ± 0.2 L, p = 0.32), and a decrease in eNO (30.4 ± 5 to 18.3 ± 4 ppb, p = 0.09) in the group with severe asthma after receiving oral prednisolone, although this did not reach significance.

We found no differences in BAL total cell counts between normal subjects and those with asthma, but the number of BAL macrophages was significantly higher in the group with moderate asthma receiving IGC treatment than in the group with mild asthma (Table 3) . The numbers of eosinophils were higher in subjects with asthma than in normal subjects, but there was no difference between the mild and moderate asthma groups (Table 3).


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  		TABLE 3. Bronchoalveolar lavage characteristics in subjects with asthma receiving ics

 
HDAC and HAT Activity in Alveolar Macrophages
Western blot analysis showed reduced expression of HDAC1 in the group of subjects with mild asthma compared with control subjects, and this difference was lost when compared with the group comprised of ICS-treated subjects with moderate asthma (Figure 1A) . There was no difference in HDAC2 (Figure 1A) or HDAC3 (data not shown) expression in normal subjects and those with asthma, and no significant correlation between the levels of HDAC activity and post-bronchodilator FEV1 or PC20.



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  		Figure 1. Activity and expression of histone deacetylases (HDAC) and histone acetyl-transferases (HAT) in bronchoalveolar alveolar lavage (BAL) macrophages, and baseline HAT activity in peripheral blood mononuclear cells (PBMC). Representative Western blot analysis of HDAC1 and HDAC2 expression in BAL macrophages isolated from 10 control subjects, 10 subjects with mild asthma and 8 subjects with asthma on inhaled corticosteroids (ICS) (A). HDAC1 expression was reduced in asthma, but restored in subjects with asthma on ICS. ß-actin was measured to control for protein loading. (B) HAT activity is reduced in alveolar macrophages from subjects with asthma compared with control subjects (***p < 0.001). Subjects with asthma on ICS do not have significantly increased HDAC activity. Numbers of subjects tested in each group are given below each bar. (C) HAT activity is increased in subjects with mild asthma compared with control subjects, and the increase is returned to normal by ICS. Numbers of subjects tested in each group are given below each bar. *p = 0.05, **p < 0.001. (D) There were no differences in HDAC activity in peripheral blood mononuclear cells (PBMCs) (closed bars) from subjects with different asthma severity and control subjects. HDAC activity was reduced in granulocytes (open bars) compared with PBMCs in all groups (***p < 0.001). Numbers of subjects tested in each group are given below each bar.

 
AM nuclear extracts from subjects with mild (n = 10) and moderate (n = 8) asthma showed reduced HDAC activity compared with healthy age-matched control subjects (n = 10) (3,031 ± 243 vs. 5,004 ± 164 arbitrary fluorescence units [AFU]/10 µg, p < 0.001). The group of patients with moderate persistent symptoms receiving ICS as a regular treatment (n = 8) showed a small increase in HDAC activity (3,540 ± 165 AFU/10 µg) compared with untreated subjects with mild asthma, but the increase was not significant (Figure 1B). HDAC1, 2, and 3 were localized to the nucleus and no obvious difference was seen in the intensity of staining (data not shown).

In contrast, total HAT activity was significantly increased in subjects with asthma not receiving ICS compared with control subjects (1.43 ± 0.1 vs. 1.01 ± 0.1 standard units/10 µg, p < 0.05), and it was reduced in the group receiving ICS (1.43 ± 0.1 vs. 0.77 ± 0.06 standard units/10 µg, p < 0.001) to normal levels (Figure 1C).

HDAC activity in PBMCs from Subjects with Asthma
We did not see any obvious differences between the activity and expression of HDACs in lymphocytes and monocytes when the methodology was being optimized, therefore, unfractionated PBMCs were used in subsequent experiments. No significant difference was observed in PBMC baseline HDAC activity in subjects with mild or moderate asthma and normal control subjects, or in different asthma severity groups (Figure 1D). Granulocytes from whole blood expressed less HDAC activity than did PBMCs (1,869 ± 40 vs. 4,789 ± 431 AFU/10 µg, respectively; p < 0.001) in both subjects with asthma and control subjects (Figure 1D).

Effect of Glucocorticoids on Cytokine Release by AMs
There were no significant differences in baseline levels of IL-8, GM-CSF, or TNF-{alpha}. Cultured AMs from subjects with asthma released more GM-CSF and TNF-{alpha} in response to LPS (Figures 2A and 2B) than control subjects (1,263 ± 124 vs. 580 ± 37 pg/ml, p < 0.001 and 4,641 ± 59 vs. 3381 ± 70 pg/ml, p < 0.001, respectively). There were no significant differences within the group comprised of subjects with asthma between those subjects with mild asthma receiving and not receiving ICS. Furthermore, LPS-induced GM-CSF release correlated inversely with the level of HDAC activity (r = –0.64, p < 0.05). This correlation failed to reach significance for TNF-{alpha} (r = –0.45, p = 0.1), and no correlation was seen for IL-8 (r = 0.02, p = 0.94). Dexamethasone (1 µM) ex vivo suppressed LPS-induced TNF-{alpha} release by 51 ± 7% in subjects with asthma (2,326 ± 73 vs. 4,641 ± 59 pg/ml) compared with 70 ± 9% in control subjects (1,020 ± 23 vs. 3,381 ± 70 pg/ml), and also suppressed LPS-induced GM-CSF by 83 ± 1% in subjects with asthma and 90 ± 1% in normal subjects. This effect was mimicked by an NF-{kappa}B inhibitor (AS602868, 10 µM). LPS-induced IL-8 release was not significantly increased in subjects with asthma compared with control subjects (74.9 ± 1 vs. 71.3 ± 2 ng/ml, p = 0.13), and dexamethasone suppression was only 20 ± 9%, which was also mimicked by the NF-{kappa}B inhibitor AS602868. The HDAC activator, theophylline (10 µM), alone had no affect on cytokine release (data not shown), but enhanced the suppressive effect of dexamethasone (1 µM) on IL-8 expression (Figure 2C), up to 37 ± 6% (16.2 ± 1.5 vs. 12.9 ± 2, p < 0.05). This effect was not significant for GM-CSF or TNF-{alpha}, in which dexamethasone (1 µM) suppression was much greater.



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  		Figure 2. Effect of dexamethasone, theophylline, and NF-{kappa}B inhibitor on cytokine release by alveolar macrophages from subjects with mild asthma (closed bars) and control subjects (open bars). Data are the mean ± SEM concentration of (A) granulocyte macrophage-colony stimulating factor (GM-CSF), (B) tumor necrosis factor-{alpha} (TNF{alpha}), and (C) interleukin-8 (IL-8) for 8 subjects in each group. *p < 0.05, **p < 0.01, and ***p < 0.00; compared with LPS-stimulation. ##p < 0.01; subjects with mild asthma compared with normal control subjects. LPS (10 µg/ml); Dex, dexamethasone (1 µM); Theo, theophylline (10 µM).

 
Effect of Prednisolone on HDAC Activity and Expression in PBMCs
Prednisolone (30 mg orally) given daily for a week significantly increased HDAC activity in PBMCs from subjects with mild asthma (4,303 ± 328 vs. 5,635 ± 457 AFU/10 µg, p < 0.05) (Figure 3A) , but not in PBMCs of subjects with severe asthma (4,666 ± 133 vs. 5,496 ± 442 AFU/10 µg, p = 0.14) (Figure 3B). Blood levels of prednisolone were higher in the group with mild asthma than in the group with severe asthma (1,588 ± 225 vs. 983 ± 127, p < 0.05). IL-8 release by PBMCs and granulocytes after 24-hour incubation was reduced after treatment with prednisolone by 79% (549 ± 109 vs. 185 ± 74 pg/ml, p < 0.05) in subjects with mild asthma and by 51% (575 ± 67 vs. 281 ± 119 pg/ml, p = 0.057) in subjects with moderate–severe asthma (Figure 3C). At the same time, prednisolone enhanced HDAC expression in PBMCs isolated from subjects with mild, but not with moderate–severe, asthma (Figure 3D).



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  		Figure 3. Effect of prednisolone on activity and expression of histone deacetylases (HDAC). Effect of prednisolone (30 mg/day for 3 weeks) on HDAC activity in peripheral blood mononuclear cells (PBMC) from subjects with (A) mild and (B) moderate–severe asthma. (C) Whole blood interleukin-8 (IL-8) release was decreased after a week course of prednisolone in subjects with mild asthma, but not with moderate–severe asthma. *p < 0.05. (D) Western blot analysis showing that expression of HDAC1 was increased in subjects with mild asthma, but not in those with moderate–severe asthma following prednisolone treatment (upper panel). Lower panel shows a graphical representation of the Western blot results relative to ß-actin loading controls. Mean and individual results are shown. Pre, pretreatment; post, posttreatment.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we found that alveolar macrophages, but not PBMCs, from subjects with asthma showed reduced HDAC and elevated HAT activity. A reduction in HDAC activity and an increase in HAT activity are associated with increased transcription of inflammatory proteins, which triggers the characteristic inflammatory cascade seen in asthmatic airways. Ito and colleagues (9) found increased HAT activity and reduced HDAC activity in bronchial biopsies from patients with mild asthma. ICS decreased HAT activity and increased HDAC activity, which together will result in suppression of NF-{kappa}B function. We have previously shown that recruitment of HDACs to the p65-HAT complex by glucocorticoids accounts for ~ 50% of their repressive actions (18). The reduced HDAC activity and the increased HAT activity found in alveolar macrophages extends our previous studies (10), and the reduction in HAT activity and the trend toward increased HDAC activity observed in the ICS-treated group is consistent with our previous findings in bronchial biopsies (9). This could underlie the increased cytokine production in response to an inflammatory stimulus, such as LPS.

In addition, we have found a difference in expression of HDAC1, but not in expression of other Class I HDACs. Furthermore, in those subjects with more severe disease receiving corticosteroid therapy, this difference was lost. This confirms previous data showing that glucocorticoids can induce HDAC expression in cell lines (7). Class II HDACs, such as HDACs 4–7 and 9, shuttle between the nucleus and the cytoplasm. Thus, subcellular localization of the isoforms, rather than expression or activity per se, is important to their actions (5). Further studies are needed to investigate the role of these HDACs. The changes seen with HDAC1 after ICS, and the reduced effect on HDAC activity after prednisolone treatment do suggest that other HDACs may be important, but which of the 17 is open to debate and beyond the scope of the present manuscript.

We found that the number of BAL macrophages was significantly higher in the subject group with moderate asthma receiving treatment with ICS compared with the subject group with mild, untreated asthma. Similar findings have been described previously. For example, Ward and colleagues (19) found a similar number of macrophages in subjects with asthma and control subjects, and this profile was not changed after 3 or 12 months of medium doses of fluticasone. This study grouped subjects with mild and moderate asthma, thus, there could be a bias of severity that may have been missed. Furthermore, Hart and coworkers (20), using similar methodology, found increased numbers of macrophages following fluticasone treatment. An unexpected finding was the presence of 1.6% of eosinophils in the BAL of subjects with moderate asthma treated with inhaled steroids. This could reflect an inappropriate treatment due to either low compliance or doses of glucocorticoids insufficient to suppress these cells.

Glucocorticoids effectively suppressed inflammatory cytokines, such as GM-CSF and TNF-{alpha} ex vivo. Recruitment of HDAC to the promoter site of NF-{kappa}B–responsive genes, along with the direct inhibition of NF-{kappa}B–associated HAT activity, is required for the full suppression of NF-{kappa}B activation by glucocorticoids (21). AS602868 has been shown to block NF-{kappa}B activation through inhibition of IKK2 in human umbilical vascular endothelial cells (16). The fact that dexamethasone suppression was identical to the effect of an NF-{kappa}B inhibitor suggests that this mechanism is important for GM-CSF and TNF-{alpha} expression. However, IL-8 was only partially suppressed through inhibition of NF-{kappa}B. This effect has been previously observed elsewhere (22). In addition, Kim and colleagues (23) described an NF-{kappa}B–independent pathway of IL-8 and IL-6 expression in human bronchial epithelial cells, and our data would support this observation. The results suggest that NF-{kappa}B is not the sole arbiter of cytokine release, and cooperative action/recruitment of other transcription factors or cofactors is required for NF-{kappa}B to regulate the transcription of different cytokines. Further evidence for this was provided by studies using chromatin immunoprecipitation showing time-dependent activation of NF-{kappa}B–dependent genes, often following AP-1 DNA binding (24, 25).

Ito and colleagues (26) described a correlation between HDAC activity and the inhibitory effect of dexamethasone on IL-8 release in alveolar macrophages of smokers (26). We increased HDAC activity ex vivo in these cells by treating them with low-dose theophylline (10), and found that theophylline enhanced the suppressive effect of dexamethasone on IL-8 release, but not GM-CSF and TNF-{alpha} release, probably because these latter two cytokines were already maximally suppressed by the high concentration of dexamethasone used in the study.

Our data show no differences in total HDAC activity in PBMCs of subjects with asthma having different degrees of severity compared with healthy control subjects. We previously studied HDAC activity of blood lymphocytes from the same group of patients and found no differences (unpublished data). Future experiments will determine whether any changes in HDAC activity in PBMCs could be related to monocytes or lymphocytes or both. Viksman and colleagues (4) have shown no differences in 17 phenotypic markers of activation in blood monocytes of normal subjects and those with asthma. These results suggest that the activation of alveolar macrophages occurs in situ within the airways and lungs, and not within the systemic circulation.

An interesting finding in our study is the very low HDAC activity observed in granulocytes (mainly neutrophils) in all subject groups. This would implicate a reduced capacity of glucocorticoids to regulate neutrophil function. This is consistent with the description by Green and coworkers (27) of a subgroup of subjects with mild–moderate asthma with predominant neutrophilic inflammation and a poor response to glucocorticoids. In our study, treatment of such subjects with prednisolone did not lead to an increase in granulocyte HDAC activity. However, prednisolone increased HDAC activity in PBMCs, but only in the group comprised of subjects with moderate asthma. In the group of subjects with severe asthma, this increase did not reach statistical significance, probably due to the effect of baseline steroid therapy and the small sample size.

In the group of subjects with mild asthma, there was a reduction of eNO after treatment, which indicates that there was an antiinflammatory effect of prednisolone in the airways that may reflect a preferential effect on HAT and HDAC activity. We perhaps expected a greater increase in HDAC activity after ICS treatment. The current data concerning primary BAL macrophages may suggest, however, that direct inhibition of HAT activity is important in macrophages. Alternatively, the group of subjects with severe asthma could have a relative insensitivity to ICS actions and, therefore, a reduced capacity to enhance HDAC activity. Ideally, we would have liked to perform a double-blind crossover study to examine the effects of glucocorticoids, but this would have required performing two bronchoscopies on subjects with severe asthma and we did not have ethical approval for this.

In summary, we have shown that one of the mechanisms of the increased inflammatory response seen in asthma may be due to a reduction in HDAC activity and an increase in HAT activity at the site of disease, but not in circulating blood cells. Glucocorticoids and theophylline can downregulate the inflammatory response, locally and systemically, by modulating HDAC and HAT activity and NF-{kappa}B activation.


   Acknowledgments
 
The authors thank Dr. Michel Dreano (Serono, Geneva, Switzerland) for the gift of AS602868.


   FOOTNOTES
 
Supported by the National Institutes of Health, the British Lung Foundation, and the Clinical Research Committee of the Royal Brompton Hospital, London. BGC was recipient of a European Respiratory Society fellowship and a Becario Separ.

Conflict of Interest Statement: B.G.C. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; B.M. does not have a financial relationship with a commercial entity that has an interest in the subject of this article; K.I. does not have a financial relationship with a commercial entity that has an interest in the subject of this article; E.J. does not have a financial relationship with a commercial entity that has an interest in the subject of this article; P.J.B. serves as a consultant to GlaxoSmithKline (GSK), is a member of scientific advisory boards for GSK, Boehringer Ingelheim, Altana, has received lecture fees from GSK, Astra Zeneca, Boehringer Ingelheim, and unrestricted grants from GSK, AstraZeneca, Boehringer Ingelheim; K.F.C. does not have a financial relationship with a commercial entity that has an interest in the subject of this article; I.M.A. has been reimbursed by GSK and Boehringer for attending several conferences, has participated as a speaker in scientific meetings or courses organized and financed by various pharmaceutical companies (GSK, MSD, Novartis, and Pfizer), and has also received research grants between 2001 and 2004 from GSK, AstraZeneca, Celgene, and ConPharma for investigating the mechanisms of glucocorticoid actions.

Received in original form May 15, 2003; accepted in final form April 9, 2004


   REFERENCES
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 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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