Upregulation of ICAM-1 expression in bronchial epithelial cells by airway secretions in bronchiectasis

ARTICLE IN PRESS Respiratory Medicine (2008) 102, 287–298

Upregulation of ICAM-1 expression in bronchial epithelial cells by airway secretions in bronchiectasis Stanley C.H. Chana, Daisy K.Y. Shuma, George L. Tipoeb, Judith C.W. Makc, Erik T.M. Leungb, Mary S.M. Ipc, a

Department of Biochemistry, Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China Department of Anatomy, Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China c Department of Medicine, Faculty of Medicine, The University of Hong Kong, Hong Kong SAR, China b

Received 11 April 2007; accepted 30 August 2007 Available online 10 October 2007

KEYWORDS Bronchiectasis; Bronchial epithelial cells; Intercellular cell adhesion molecule; Tumor necrosis factor-a; Drug modulation

Summary The airway epithelium participates in chronic airway inflammation by expressing adhesion molecules that mediate the transmigration of neutrophils into the inflamed airways. We hypothesize that, in patients with bronchiectasis, cytokines in their bronchial secretions enhance the expression of intercellular cell adhesion molecule (ICAM-1) in the bronchial epithelium and thus contribute to sustained recruitment of neutrophils into the inflamed airways. In the present study, we investigated the effect of bronchial secretions on the regulation of ICAM-1 in bronchial epithelial cells, and its modulation by pharmacologic agents. The expression of ICAM-1 mRNA and protein in human bronchial epithelial cells upon exposure to sputum sol from subjects with bronchiectasis were evaluated by reverse transcription-polymerase chain reaction (RT-PCR) and ELISA, respectively. Modulating effects of dexamethasone, ibuprofen, MK-663 or triptolide on ICAM-1 regulation were investigated in vitro. We demonstrated that changes in ICAM-1 expression correlated with levels of TNF-a in the sputum sol, and treatment of sol samples with TNF-a antibodies attenuated both the increase in ICAM-1 mRNA and protein. The role of TNF-a was further demonstrated when TNF-a elicited dose dependent increase in ICAM-1 expression. The sputum effect could also be suppressed dose-dependently by pre-incubation of bronchial epithelial cells with dexamethasone, ibuprofen, MK-663 or triptolide. Evidence is thus provided for the upregulation of bronchial epithelial ICAM-1 expression by airway

Abbreviation: BEGM, bronchial epithelial cell growth medium; ELISA, enzyme-linked immunosorbent assay; ICAM-1, intercellular cell adhesion molecule; IL-1b, interleukin 1b; IL-6, interleukin 6; IL-8, interleukin 8; NHBE, normal human bronchial epithelial cells; TNF-a, tumor necrosis factor-a Corresponding author. Division of Respiratory and Critical Care Medicine, Department of Medicine, The University of Hong Kong, Queen Mary Hospital, Pokfulum, Hong Kong SAR, China. Tel.: +852 2855 4605; fax: +852 2816 2863. E-mail address: [email protected] (M.S.M. Ip). 0954-6111/$ - see front matter & 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.rmed.2007.08.013

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Introduction

Subjects and sputum samples processing

Bronchiectasis is characterized by irreversibly dilated bronchi that are chronically inflamed and often colonized by bacteria. Intense and persistent neutrophil recruitment to the airways and the release of neutrophil elastase are hallmarks in the pathogenesis of bronchiectasis.1–5 Recruitment of neutrophils to the airways depends on a stepwise process which allows neutrophils to transmigrate across the vascular endothelium, airway epithelium and eventually into the airway lumen. Activation and adhesion of neutrophils onto bronchial epithelial cells is regulated by a cytokine cascade and specific adhesion molecules.6–8 Among various adhesion molecules, intercellular cell adhesion molecule-1 (ICAM-1), a transmembrane glycoprotein of 505 amino acids, mediates the interaction between the neutrophils and bronchial epithelial cells.9,10 ICAM-1 is constitutively expressed on endothelium, epithelium and neutrophils, and its upregulation is triggered by a number of inflammatory mediators, including interferon-g, interleukin1b (IL-1b) and tumor necrosis factor-a (TNF-a).11,12 Upregulated expression of ICAM-1 has been demonstrated in patients with asthma, COPD and bronchiectasis.7,13,14 Increased expression of alveolar epithelial ICAM-1 was reported to enhance adherence of neutrophils and macrophages in vivo and in vitro,15 suggesting a biological function of ICAM-1 in the perpetuation of bronchial injury. This study was designed to determine the effects of secretions from inflamed airways on ICAM-1 expression in bronchial epithelium, specifically the role of TNF-a. The study further explored the use of pharmacologic agents, including antibody against TNF-a, glucocorticoid, cox-1 and cox-2 inhibitors, and triptolide, a Chinese herbal extract, in modulating the sputum effect.

Patients with bronchiectasis were recruited from the Bronchiectasis Clinic, The University of Hong Kong, Queen Mary Hospital. Inclusion criteria were: bronchiectasis documented on high resolution computerized tomography (HRCT) of chest; idiopathic etiology of bronchiectasis; chronic sputum production with daily sputum 410 ml; absence of asthma (according to American Thoracic Society guidelines)16 and other major pulmonary diagnoses; and a steady state as defined by absence of change of symptoms noted by the patient over the past 3 weeks. Exclusion criteria were: bronchiectasis with defined etiology (e.g. post-tuberculous, primary ciliary dyskinesia, common variable immunodeficiency); maintenance use of oral or nebulized antibiotics; use of antibiotics within the previous 3 weeks. A sweat test has not been performed to exclude cystic fibrosis because of the known rarity of cystic fibrosis among Chinese and the lack of suggestion of multi-system disease in any of the patients. Lung function testing was done using SensorMedics 2200 (SensorMedics, Yorba Linda, CA) lung function system. Sputum samples were collected over a maximum of 4 h in sterile pots. Patients taking inhaled bronchodilators or inhaled corticosteroids have been advised to omit these drugs for at least 12 h before sputa were collected. Healthy subjects who were asymptomatic of respiratory diseases and have not been receiving regular medications were recruited randomly from laboratory personnel. Sputum induction in healthy volunteers was performed using a method as described by Keatings et al.17 Induced sputum from healthy subjects were used as control for studying the effect of sputum sol from bronchiectasis subjects on ICAM-1 expression in bronchial epithelial cells. Expectorated sputum from both patients and control subjects were examined under light microscopy and presence of X10 buccal squamous epithelial cells per field (  100 magnification), as average of 10 fields, being considered to indicate a salivary origin, and samples were excluded from further processing. Sputum samples from patients were also sent for microbiological examination using standard techniques. The bulk of the collected sputum samples were immediately centrifuged at 50,000g for 1.5 h (4 1C); the supernatant sol phase of each sample was collected, aliquoted and stored at 80 1C until use. The study has been approved by The University of Hong Kong Ethics Committee; patients and healthy volunteers gave written informed consent before sputum collection.

Methods Reagents and antibodies Dexamethasone, ibuprofen and nimodipine were products of Sigma (St. Louis, MO, USA) and MK-663 (COX-2 inhibitor) was obtained from the Merck Research Laboratory. Triptolide was kindly provided by Professor DQ Yu (Chinese Academy of Medical Sciences, Beijing, China). Dexamethasone and ibuprofen were prepared in methanol, MK-663 was prepared in distilled water and triptolide was prepared in ethanol/acetone (v/v, 1:1), nimodipine was first dissolved in DMSO and then diluted in cell culture medium to the working concentration. Recombinant human tumor necrosis factor-a (rh TNF-a), anti-human TNF-a and antihuman IL-1b were obtained from PeproTech EC Ltd. (London, UK).

Primary culture of human bronchial epithelial cells Normal human bronchial epithelial cells (NHBE) (Clonetics, San Diego, CA, USA) were cultured in bronchial epithelial cell growth medium (BEGM) (Clonetics) containing bovine

ARTICLE IN PRESS Upregulation of ICAM-1 expression in bronchiectasis pituitary extract (30 mg/ml), hydrocortisone (0.5 mg/ml), human epidermal growth factor (0.5 ng/ml), epinephrine (0.5 mg/ml), transferrin (10 mg/ml), insulin (5 mg/ml), retinoic acid (1 ng/ml), triiodothyronine (6.51 ng/ml), gentamicin (50 mg/ml) and bovine serum albumin (500 mg/ml) at 37 1C and 5% CO2. Cell viability was checked with trypan blue exclusion test to ensure no significant difference between various experiments and to ascertain that cells were viable after incubation with the high concentrations of drugs used in the study.

Assay of TNF-a level in sputum sol The protein level of TNF-a in the sputum sol phase was measured with the use of a quantitative sandwich enzymelinked immunosorbent assay (ELISA) kit (PeproTech, London, UK) as described by Shum et al.8 Samples (100 ml) of diluted sputum sol were dispensed into wells of microtiter plates which had been coated with monoclonal anti-human TNF-a. After incubation for 3 h at room temperature, the wells were first washed with phosphate-buffered saline (PBS) and then incubated with an enzyme-linked polyclonal rabbit antibody directed against TNF-a for another 45 min at room temperature. After further rinses with PBS to remove the unbound antibodies, substrate solution (40 ml) was added to each well and incubated for 20 min at 37 1C. The reaction was terminated with the addition of a ‘‘stop’’ solution (0.5 M sulfuric acid, 100 ml). Assays were performed in duplicate. Absorbance was measured at 492 nm in an ELISA reader. Values were determined relative to a standard curve.

Quantification of NHBE expression of ICAM-1 protein The level of ICAM-1 expression was determined by ELISA as described previously.18 NHBE cells (1  105 cells per well) were washed twice with PBS and fixed at room temperature with 4% paraformaldehyde for 10 min. After washing with PBS, they were blocked with 1% BSA in Tris-buffered saline containing 0.05% Tween 20 for 15 min. The cells were then incubated sequentially with mouse anti-human ICAM-1 antibody (primary antibody, diluted 1:100; Chemicon, CA, USA) for 1 h and horseradish peroxidase-labeled anti-mouse antibody (secondary antibody, 1:1000, serotec, oxford, UK) for 30 min. OPD substrate (0.4 mg/ml in phosphate-citrate buffer, pH 5.0; 24.3 mM citric acid, 51.4 mM Na2HPO4  12H2O, 12% H2O2, v/v) was then applied to the cells for 30 min and sulfuric acid (3 M) was added to stop the reaction. Each assay was performed in triplicate. Absorbance was measured at 450 nm in an ELISA reader and corrected for background by subtracting the absorbance of controls in which the primary antibody had been omitted.

Treatment of NHBE cells with sputum sol, anti-TNFa, anti-IL-1b or rh-TNF-a Sputum sol samples (100 ml) were incubated with NHBE cell cultures at 37 1C and 5% CO2 for 8 h. To prevent cell detachment by the action of sputum elastase and metalloproteinases, the culture medium was supplemented with eglin C (0.05 M; a specific inhibitor of neutrophil elastase, Sigma), and EDTA (0.05 M). Treatments were performed in

289 triplicate for each sputum sol sample. Parallel incubations of NHBE cells with two induced control sputum samples were performed. The cells were then assessed for ICAM-1 mRNA expression by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) and for ICAM-1 protein by ELISA. To neutralize the effects of sputum sol TNF-a and IL-1b on the NHBE cells, sputum sol samples were preincubated (4 1C) with anti-TNF-a or anti-IL-1b for 1 h prior to the incubation with NHBE cells for 8 h. Alternatively, rh-TNFa was added to NHBE cell cultures at concentrations of 50–1000 pg/ml and incubated for 8 h. ICAM-1 mRNA and protein expression was assessed by semi-quantitative RT-PCR and ELISA, respectively.

Treatment of NHBE cells with anti-inflammatory drugs To evaluate the potential effect of various anti-inflammatory drugs on ICAM-1 mRNA expression, the NHBE cells were first treated with dexamethasone, ibuprofen, MK-663, triptolide or nimodipine for 1 h prior to the stimulation with sputum sol samples for 8 h. The drug concentrations were chosen on the basis of data from previously published studies19–22 or from the supplier (Merck Research Laboratory for MK-663; Sigma for nimodipine). Incubations of NHBE cells was performed with two control sputum simultaneously. ICAM-1 mRNA and protein expression was assessed by semiquantitative RT-PCR and ELISA, respectively.

Assay with actinomycin D and cycloheximide NHBE cells were first treated with either 5 mg/ml of actinomycin D (Sigma) or 50 mg/ml cycloheximide (Sigma) for 1 h prior to the stimulation with sputum sol samples for 8 h. ICAM-1 mRNA expression was assayed semi-quantitatively with RT-PCR and the level of ICAM-1 protein expression was determined by ELISA as described.

RT-PCR analysis of ICAM-1 mRNA expression Total RNA was extracted from the cells by the RNeasys Mini kit (Qiagen, Valencia, CA, USA). The extracted RNA samples were quantified by measurement of absorbance at 260 nm using GeneQuantTM (Amersham Biosciences, Piscataway, NJ, USA). RNA (2 mg) from each sample was reversed transcribed into cDNA with random oligo (dT) primers and SuperScriptTM II reverse transcriptase (Invitrogen, Carlsbad, CA, USA). Equal amounts of the cDNA were amplified with PCR in a final volume of 20 ml. The primers for amplifying ICAM-1 cDNA were: forward 50 -TATGGCAACGACTCCTTCT-30 and reverse 50 -CATTCAGCGTCACCTTGG-30 . The primers for amplifying glyceraldehyde phosphate dehydrogenase (GAPDH) cDNA were: forward 50 -CCTTCATTGACCTCAACTACATGGT-30 and reverse 50 -TCGTTGTCATACCAGGAAATGAGCT-30 . The PCR reagents and Taq polymerase were purchased from (Invitrogen). Amplification was started with denaturation at 95 1C (12 min) followed by 30 cycles for ICAM-1 and 25 cycles for GAPDH. Each cycle consisted of 2 min at 95 1C, 30 s at 55 1C (for GAPDH, 1 min at 60 1C) and 90 s at 72 1C. The final extension was 10 min at 72 1C. After amplification, PCR products (5 ml each) were separated by electrophoresis in a

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1% (w/v) agarose gel containing ethidium bromide (0.5 mg/ml); results were visualized under ultraviolet light. The PCR signals were quantified using the Image J quantification software (National Institute of Health, USA). For each sample, ICAM-1 signal was normalized against that of the house-keeping gene, GAPDH and expressed as signal ratios of ICAM-1/GAPDH.

Statistical analysis Data were expressed as mean7S.D. The INSTAT and PRISM statistical software packages (GraphPad Inc., San Diego, CA) were used for the statistical analyses. Data were analyzed by one-way analysis of variance (ANOVA) with a Bonferroni’s post hoc test. Correlation between ICAM-1 protein expression in NHBC cells and sputum TNF-a level in the culture medium was determined with Spearman’s rank correlation test. A P-value of o0.05 was considered statistically significant.

Results Clinical characteristics This study recruited 12 subjects with idiopathic bronchiectasis proven by HRCT scan of thorax and 6 healthy volunteers as controls. Their demographic and clinical features are shown in Table 1.

Sputum samples of patients with bronchiectasis stimulate NHBE expression of ICAM-1 The level of ICAM-1 mRNA in the primary cultures of NHBE cells was increased with exposure to sputum sol samples of 12 patients with bronchiectasis. Test samples of sputum sol elicited a 1.7-fold increase in ICAM-1 mRNA expression when compared with the six induced sputum controls (0.7370.10 vs. 0.4270.03, ICAM-1/GAPDH ratio) (Figure 1a). Similarly, exposure of cultures of NHBE cells to sputum sol samples elicited a 1.8-fold increase in ICAM-1 protein levels when compared with the six induced sputum controls (0.8570.10 vs. 0.4770.03) in terms of absorbance at 450 nm) (Figure 1b).

TNF-a is a key sputum cytokine that stimulates NHBE expression of ICAM-1 NHBE expression of ICAM-1 protein was found to correlate with the level of TNF-a as presented by sputum sol samples in the NHBE culture medium (r ¼ 0.9441, Po0.001, Figure 2). Pre-incubation of sputum sol samples with commensurate level of neutralizing antibody against TNF-a resulted in significant reduction of both ICAM-1 mRNA and protein expression in contrast to the insignificant reduction of ICAMexpression in anti-IL-1b-treated samples (Figure 3a and b). Treatment of NHBE cells with rh-TNF-a resulted in dosedependent significant increase in both ICAM-1 mRNA and protein expression (Figure 4a and b).

Effect of actinomycin D and cycloheximide on sputum sol induced ICAM-1 expression Table 1

Characteristics of study subjects.

Sex (M:F) Age (year)

Patients with bronchiectasis

Healthy controls

6:6 48717

4:2 2874

Sputum microbiology profile Pseudomonas 3 aeruginosa Hemophilus 1 influenzae Commensals 8

N/A

Lung function test results FEV1 (L) 1.270.7 FVC (L) 2.270.7

Not measured Not measured

Smoking habit Non-smoker

12 (100%)

6 (100%)

3 (2–5)

N/A

HRCT thorax No. of lobes affectedy

N/A: not applicable.  Values are expressed as mean7S.D. y Values are expressed as median (range).

N/A

Pretreatment of the NHBE cells with a RNA polymerase inhibitor, actinomycin D, resulted in a decrease in ICAM-1 mRNA level while cycloheximide, a protein synthesis inhibitor, did not inhibit upregulation of ICAM-1 at mRNA level (Figure 5a). On the other hand, both actinomycin D and cycloheximide inhibited ICAM-1 protein expression (Figure 5b).

Effect of dexamethasone, ibuprofen, MK-663 and triptolide on ICAM-1 expression

N/A

Pretreatment of NHBE cells with 0.2 mM dexamethasone decreased ICAM-1 expression but this was not statistically significant (P ¼ 0.065). However, higher concentrations of dexamthasone (2–20 mM) resulted in dose-dependent significant decrease in ICAM-1 mRNA and protein expression when compared with the sputum sol-stimulated NHBE cells (Figure 6a and b). Similarly, pretreatment of the NHBE cells with MK-663 (2–200 mM) or iburopfen (2–200 mM) resulted in dose-dependent down-regulation of ICAM-1 expression when compared with the sputum sol-stimulated NHBE cells (Figure 6a and b). On the other hand, triptolide was more potent than the other anti-inflammatory agents tested in our study. Pretreatment of the NHBE cells with much lower concentrations (0.02–2 mM) of triptolide dose-dependently down-regulated ICAM expression to level achieved with the other drugs

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Figure 1 The effect of sputum sol samples from patients with bronchiectasis on NHBE expression of ICAM-1. (a) Total RNAs were extracted from the NHBE cells after incubation with sputum sol samples of patients with bronchiectasis or induced sputum of healthy volunteers and analyzed for ICAM-1 mRNA expression by RT-PCR. The top panel shows gel bands due to PCR amplification products of ICAM-1 (238 bp) and GAPDH (844 bp) transcripts. ICAM-1 mRNA was assessed by integration of pixel density of the band and expressed as a ratio to that of GAPDH mRNA. (b) NHBE expression of ICAM-1 protein as a result of incubation with sputum sol samples of patients with bronchiectasis or induced sputum of healthy volunteers. ICAM-1 protein was determined with ELISA and expressed in terms of absorbance at 450 nm, net that of background. The data, shown as mean7S.D., were derived from 12 sputum samples from patients with bronchiectasis and 6 induced sputum samples from healthy volunteers, each tested in triplicate. ***Po0.001.

(Figure 6a and b). Nimodipine, a calcium channel blocker, serve as a negative control in our experiment. Pretreatment of NHBE cells with nimodipine showed no significant effect on ICAM-1 expression in sputum sol-stimulated NHBE cells (Figure 6a and b). The inhibitory effect on ICAM-1 expression was not related to toxicity of these drugs since epithelial cells remained 98% viable as determined by the Trypan blue dye exclusion test at the end of the culture periods. In addition,

pre-incubation of NHBE cells with drug vehicles (methanol, ethanol, acetone and DMSO) showed no significant effect on ICAM-1 expression (results not shown).

Discussion The major findings in this study were that bronchial secretions of patients with idiopathic bronchiectasis were

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ICAM-1 protein level (absorbance 450 nm)

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Figure 2 Correlation between NHBE expression of ICAM-1 protein and TNF-a level as presented by bronchiectasis patient sputum sol samples in the culture medium. Sputum sol samples (100 ml each) were diluted 1-in-10 (v/v) in NHBE culture media during the 8-h incubation. Accordingly, the concentration of TNF-a measured by ELISA in 100 ml of undiluted sputum was pro-rated to that in the medium for the plot against NHBE expression of ICAM-1. Spearman’s correlation coefficient (r) and its significance (P) are indicated.

active in upregulating expression of ICAM-1 mRNA and ICAM-1 protein by human bronchial epithelial cells in vitro, and that TNF-a in the sputum sol samples was responsible for this effect. Apart from the neutralizing effect of anti-TNF-a antibody, pharmacologic agents including dexamethasone, ibuprofen, MK-663 and triptolide were shown to counteract the sputum effect on ICAM-1 upregulation. Neutrophil recruitment represents an important event of host response to injury, although the end result may not always be favorable to host tissues. The pathogenesis of bronchiectasis has been shown to involve persistent recruitment of neutrophils from the circulation into the bronchial lumen with subsequent release of enzymes leading to airway damage.1–5 ICAM-1, a member of the immunoglobulin superfamily, is one of the important components that play critical roles in neutrophil recruitment and adhesion towards the damaged tissues.23 ICAM-1 upregulation can be triggered by extravascular bacterial products and pro-inflammatory mediators derived from the epithelium.24,25 Both in vivo and in vitro studies have demonstrated that bacterial lipopolysaccharide and pro-inflammatory cytokines including IL-1b, TNF-a and IFN-g upregulated the expression of ICAM-1 in airway epithelial cells.9,11,12 High levels of IL-1b, TNF-a, and IL-8 have been consistently found in the expectorated bronchial secretions of patients with bronchiectasis.2,26 TNF-a has been reported to play important roles in neutrophilmediated degradation of lung matrix components in bronchiectasis8 and induction of the expression of IL-827 and IL-6.28 In this study, we demonstrated that TNF-a in bronchial secretions of patients with bronchiectasis is a major mediator in the upregulation of ICAM-1 expression in human bronchial epithelial cells. There has been great interest in the use of various antiinflammatory drugs to overcome chronic inflammation cascade in disease. The key roles of TNF-a in initiating

inflammatory and maintaining cellular immune responses have led to the development of new therapies. Anti-TNF-a agents including infliximab (Remicades), etanercept (Enbrels) and adalimumab (Humiras) have demonstrated efficacy in the treatment of rheumatoid arthritis29 and inflammatory bowel disease.30 However, as non-selective blockade of TNF may well result in increased infections, and there has been reports of increased risk of reactivation of tuberculosis in subjects treated with anti-TNF-a.31 In particular, bronchiectasis is a condition with persistent microbial colonization, the risk of worsening the infective component cannot be over-emphasized.32 The in vitro antagonistic effect of anti-TNF antibody on NHBE upregulation of ICAM-1 confirmed the role of TNF-a as an inflammatory mediator in bronchiectasis, although the use of antiTNF-a agent may not be beneficial to the patients of bronchiectais. Dexamethasone is a potent steroid widely used in treatments of inflammatory conditions. Oral prednisolone has been reported to improve clinical parameters in children with cystic fibrosis,33 but side-effects of long-term use prohibit its clinical utility. Inhaled steroids have been shown to down-regulate inflammatory mediators in bronchiectasis.26 However, a randomized trial of inhaled fluticasone only showed a decrease in sputum volume but no change in FEV1 or frequency of exacerbation.34 Previous studies have shown that 0.1 mM dexamethasone can effectively down-regulate TNF-a and rhinovirus-induced ICAM-1 expression in both human nasal epithelial cells35 and A549 type II pulmonary cells.36 In our study, 0.2 mM dexamethasone did not significantly inhibit ICAM-1 expression, while higher concentrations (2 and 20 mM) showed definite inhibitory effects. This suggests that there may be other factors in the sputum sol that can counteract the action of dexamethasone on ICAM-1 expression. The in vitro findings support the advantageous edge of using inhalational route of administration

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Figure 3 Anti-TNF-a counteracted sputum sol stimulation of NHBE expression of ICAM-1. Sputum sol samples had been preincubated with either anti-TNF-a (5 ng/ml) or anti-IL 1b (5 ng/ml) 1 h prior to incubation with NHBE cells. (a) ICAM-1 mRNA data are expressed in terms of ratios (ICAM-1:GAPDH) of pixel densities due to the respective PCR products, and plotted as % relative to that of control incubations. (b) ICAM-1 protein levels are expressed in terms of absorbance at 450 nm, net that of background. The net absorbance was plotted as % of that due to control incubations. The data, shown as mean7S.D., were derived from 12 sputum sol samples, each tested in triplicate.

in vivo as this can achieve much higher concentrations in the airway epithelium, giving a more acceptable effect–side effect profile compared to systemic steroids. Clinical studies have demonstrated the use of NSAID and selective COX-2 inhibitors to attenuate the inflammatory

response. Inhalation of indomethacin was found to reduce the sputum volume in patients with chronic bronchitis and bronchiectasis.37 Systemic administration of indomethacin however resulted in no improvements in sputum albumin, elastase and myeloperoxidase levels.38 Ibuprofen was

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Figure 4 NHBE upregulation of ICAM-1 expression as a result of stimulation with recombinant TNF-a. Dose-dependent increase of ICAM-1 expression in NHBE cells as elicited by recombinant TNF-a. NHBE cells were treated with recombinant TNF-alpha (50, 200 and 1000 pg/ml). (a) ICAM-1 mRNA data are expressed in terms of ratios (ICAM-1:GAPDH) of pixel densities due to the respective PCR products. The ratios were plotted as %, relative to that of null treatment. (b) ICAM-1 protein levels are expressed in terms of absorbance at 450 nm, net that of background. The net absorbances were plotted as % of that due to null treatment. The data, shown as mean7S.D., were derived from five independent experiments, each performed in triplicate. Po0.01, Po0.001.

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Figure 5 The effect of ACTD and CHX on ICAM-1 mRNA and protein expression in sputum sol stimulated NHBE cells. Lack of increase in ICAM-1 mRNA and protein level when sputum sol-stimulated NHBE cells was treated with actinomycin D and cycloheximide, respectively. (a) ICAM-1 mRNA data are expressed in terms of ratios (ICAM-1:GAPDH) of pixel densities due to the respective PCR products. The data are plotted as %, relative to that of control incubations. (b) ICAM-1 protein levels are expressed in terms of absorbance at 450 nm, net that of background. The net absorbances were plotted as % of that due to control incubations. The data, shown as mean7S.D., were derived from five independent experiments, each tested in triplicate. Po0.001 when compared with NHBE cultures that were stimulated with sputum sol.

reported to improve clinical manifestations of bronchiectasis in cystic fibrosis,33 while in vitro studies showed that ibuprofen and cyclooxygenase-2 inhibitors down-regulated cytokine-induced ICAM-1 expression.37,39 Our findings support that these agents may be able to interrupt the inflammatory cycle in bronchiectasis. The use of triptolide in the treatment of inflammatory diseases has largely been based on extracts of the Chinese herb, Tripterygium wilfordii. Although oral doses of several hundred milligrams of extract per day appeared to be safe and beneficial to patients with rheumatoid arthritis, it is

unclear how these compare with nanomolar triptolide delivered to environments of cells in culture. It is however noteworthy that a 20–140 nM range was used effectively to interfere with expression of pro-inflammatory cytokines, proMMPs and to upregulate TIMPs in IL-1-treated synovial fibroblasts.40 Incidentally, this approximated the IC50 of 50–140 nM for down-regulation of pro-inflammatory cytokines as stimulated by phorbol ester, TNF-a or IL-1b treatment of human bronchial epithelial cells in culture.22 Thus, anti-inflammatory efficacy may be comparable at the cell level but clinical dosing may well differ on account of

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Triptolide

Nimodipine

Figure 6 Inhibitory effects of anti-inflammatory drugs on sputum sol stimulation of NHBE expression of ICAM-1. Dexamethasone (0.20, 2 and 20 mM), ibuprofen (2, 20 and 200 mM), MK-663 (2, 20 and 200 mM) and triptolide (0.02, 0.20 and 2 mM) showed dosedependent inhibition of the sputum effect on ICAM-1 expression. Nimodipine (2, 20 and 200 mM), a calcium channel blocker, serve as a negative control. (a) ICAM-1 mRNA data are expressed in terms of ratios (ICAM-1:GAPDH) of pixel densities due to the respective PCR products. The data are plotted as %, relative to that of control incubations. (b) ICAM-1 protein levels are expressed in terms of absorbance at 450 nm, net that of background. The net absorbances were plotted as % of that due to control incubations. The data, shown as mean7S.D., were derived from 12 sputum sol samples, each tested in triplicate. Po0.05, Po0.01, Po0.001 when compared with NHBE cultures that were stimulated with sputum sol but had not been treated with drugs.

the route of delivery and accessibility of the target at the tissue/organ level. In reports of the use of triptolide in rheumatoid arthritis, toxicity is a major limitation in its clinical application,41 particularly toxicity in the renal and cardiac systems.42

In the present study, we demonstrated upregulation of ICAM-1 protein expression accompanied by increase in ICAM-1 mRNA, suggesting regulation of gene expression at the transcriptional level in NHBE cells. This suggestion was confirmed by observations of lack of increase in basal ICAM-1

ARTICLE IN PRESS Upregulation of ICAM-1 expression in bronchiectasis mRNA expression and protein expression despite exposure to sputum sol, when actinomycin D and cycloheximide were included in NHBE cultures, respectively. Signal transduction pathways involved in the activation of ICAM-1 gene expression were found to include protein kinase C, MAP kinase and the nuclear factor NF-Kappa B signaling cascade.24,43 The promoter region of human ICAM-1 consists of not only the glucocorticoid response element (GRE) and the interferonstimulated response element (ISRE) but also a putative recognition sequence of NF-Kappa B.44 Among these, the NFKappa B family proteins appear to be essential for enhancing ICAM-1 gene expression. Deletion of NF-Kappa B promoter sequences resulted in reduction of the rhinovirus-induced ICAM-1 expression in A549 type II pulmonary cells.45 Ibuprofen can inhibit the activation and translocation of NF-Kappa B into the nucleus,46,47 while the molecular mechanism of dexamethasone in inhibiting gene expression may be mediated through the interaction between the activated glucocorticoid receptor and NF-Kappa B.48,49 Moreover, a recent study has shown that triptolide is a potent inhibitor of the NF-Kappa B signaling pathway.22 It is thus conceivable that down-regulation of ICAM-1 gene expression by dexamethasone, ibuprofen, MK-663 or triptolide is mediated through the NF-Kappa B signaling pathway.

Conclusion In summary, the study has provided evidence that bronchial secretions in bronchiectasis is capable of stimulating ICAM-1 expression in bronchial epithelial cells, and that TNF-a in the secretions is an important mediator. Apart from anti-TNF antibody, several pharmacologic agents can mitigate this upregulation in vitro. The findings provide further insights into the pathogenetic mechanisms in bronchiectasis and the potential development of therapies in the management of this disease.

Conflict of interest There is no conflict of interest for the data presented in this manuscript.

Acknowledgment This work was supported by the Hong Kong Research Grants Council (Grant no. 7286/99 M).

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