Comparative effects of inhaled budesonide and the NO-donating budesonide derivative, NCX 1020, against leukocyte influx and airway hyperreactivity following lipopolysaccharide challenge

Pulmonary Pharmacology & Therapeutics 17 (2004) 219–232 www.elsevier.com/locate/ypupt

Comparative effects of inhaled budesonide and the NO-donating budesonide derivative, NCX 1020, against leukocyte influx and airway hyperreactivity following lipopolysaccharide challenge Barra J. Nevin, Kenneth J. Broadley* Division of Pharmacology, Welsh School of Pharmacy, Cardiff University, Cathays Park, Cardiff CF10 3XF, UK Received 27 June 2003; accepted 7 April 2004

Abstract Lipopolysaccharide (LPS) inhalation (30 mg ml21, 1 h) caused airway hypereactivity (AHR) to histamine (1 mM, 20 s) 1 h later in conscious guinea-pigs. Bronchoalveolar lavage fluid (BALF) levels of neutrophils, myeloperoxidase (MPO) and protein were elevated whereas nitric oxide (NO) metabolites were reduced 1 h after LPS compared with saline challenge. 24 h after LPS, there was no AHR, but BALF neutrophils, eosinophils, macrophages, MPO, protein and NO metabolites were all raised. Budesonide (0.7 mM) and a molar equivalent concentration of the NO-donating budesonide derivative, NCX 1020, were inhaled (15 min) at 24 h and 45 min before LPS. The only change produced by budesonide was to reduce eosinophil influx at 24 h after LPS, compared with vehicle treated animals. NCX 1020, however, blocked AHR and reduced neutrophils (1 and 24 h) and MPO (1 and 24 h), while NO levels were raised at 1 and reduced at 24 h after LPS. The combined inhalation before LPS of the NO donor, SNAP (1.4 mM), with budesonide (0.7 mM) blocked the AHR to histamine and significantly reduced neutrophils (1 and 24 h) and MPO (1 and 24 h), while NO levels were raised at 1 h after LPS. Thus, NO and a corticosteroid co-administered as NCX 1020 or budesonide with a NO donor, have an additive effect against LPS-induced inflammatory responses and may have value in the treatment of neutrophil-driven airways disease such as COPD. q 2004 Elsevier Ltd. All rights reserved. Keywords: Corticosteroid; Lipopolysaccharide; Neutrophil; Nitric oxide; NCX 1020

1. Introduction Chronic obstructive pulmonary disease (COPD) is characterised by airflow obstruction and airway hyperreactivity (AHR). It is the sixth leading cause of death and the 12th leading cause of morbidity worldwide [1]. The neutrophil plays a central role in the pathological features Abbreviations: AHR, airway hyperreactivity; AP-1, activator protein 1; BALF, bronchoalveolar lavage; cNOS, constitutive nitric oxide synthase; COPD, chronic obstructive pulmonary disease; eNOS, endothelial nitric oxide synthase; FEV1, Forced Expiratory Volume in 1 second; GMP, guanosine 30 , 50 cyclic monophosphate; IFNg, interferon g; IL1b, Interleukin 1b; iNOS, inducible nitric oxide synthase; LPS, lipopolysaccharide; MPO, myeloperoxidase; NF-kB, nuclear factor-kB; nNOS, neuronal nitric oxide synthase; Gaw, specific airways conductance; SNAP, S-nitroso-Nacetylpenicillamine; TNFa, tumour necrosis factor a. * Corresponding author. Tel.: þ44-29-2087-5832; fax: þ 44-29-20874149. E-mail address: [email protected] (K.J. Broadley). 1094-5539/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.pupt.2004.04.002

of COPD [2]. Increased numbers of neutrophils in bronchoalveolar lavage fluid (BALF) in COPD have been shown to be associated with reduced airway function in COPD [3]. Unlike asthma, corticosteroids are ineffective in reducing inflammation and do not always improve symptoms in COPD [4,5]. The terminal guanidine nitrogen atom(s) of L -arginine are the physiological precursors of endothelium-derived NO. The endothelial nitric oxide synthase (eNOS), also known as NOS-3, is Ca2þ/calmodulin-dependent and is a constitutive enzyme (cNOS). cNOS and/or its isoforms may also be found in neuronal (nNOS), also known as NOS-1 and epithelial cells. The cNOS enzyme produces only picomolar quantities of NO. An inducible nitric oxide synthase (iNOS), also known as NOS-2, is found in neutrophils, eosinophils, macrophages and epithelial cells. Unlike cNOS, it produces nanomolar quantities of NO and is Ca2þ/calmodulin independent. It is induced by cytokines

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(interleukin 1b (IL1b), tumour necrosis factor a (TNFa), interferon g (IFNg)) and lipopolysaccharide/endotoxin (LPS) and its induction is inhibited by glucocorticosteroids [6]. Inhaled NO passes readily into smooth muscle to activate soluble guanylate cyclase, resulting in increased guanosine 30 , 50 cyclic monophosphate (cyclic GMP) to produce bronchodilatation and as a consequence opposes any AHR to spasmogens [7]. Exogenous NO has been found to inhibit chemotaxis, adhesion, degranulation, leukotriene production and superoxide production from neutrophils in vitro and therefore to be anti-inflammatory [8]. In ischaemia/reperfusion injury of distal microvascular beds in the cat, inhaled NO was shown to reduce oxidant-dependent leukocyte rolling, adhesion and migration and endothelial dysfunction [9]. In contrast, however, iNOS-derived NO by reacting with superoxide, may form peroxynitrite which may contribute to AHR, oedema and inflammation [6]. Increased exhaled NO levels in COPD were found to be inversely correlated with forced expiratory volume in 1 s (FEV1) and other markers of lung function [10]. Similarly, an inverse correlation between nitrotyrosine staining (a measure of peroxynitrite) in sputum inflammatory cells and FEV1 in COPD patients has been reported [11]. It appears, therefore that iNOS-derived NO in COPD may be deleterious. It has been demonstrated, however, that administration of further NO in the form of inhaled NO does not deleteriously affect pulmonary function and symptoms in COPD [12]. Therefore, in the lung NO may on the one hand be pro-inflammatory and contribute to AHR but on the other hand be bronchodilatatory and exert anti-inflammatory effects. NO metabolite levels (nitrate and nitrite) and neutrophil numbers increase in BALF 24 –48 h following LPS exposure in the guinea-pig. Specific airways conductance does not change following LPS but AHR to histamine occurs 1 h following LPS exposure and is associated with a deficiency of NO. Since there is an increase in neutrophil influx, induction of iNOS and impairment of lung function, this system may be used as a model for COPD [13]. In the present study, the NO donor, S-nitroso-Nacetylpenicillamine (SNAP) and the corticosteroid, budesonide, were used as comparators to investigate the synergistic or additive effects of the NO and steroid components of the novel NO-donating budesonide derivative (NCX 1020) in modulating AHR 1 h following LPS exposure and neutrophil influx 1 and 24 h following LPS exposure. Neutrophil myeloperoxidase (MPO) and protein levels were measured in BALF as a measure of neutrophil activation [14] and increased plasma exudation [15], respectively. The total NO metabolite levels (nitrate and nitrite) were measured in BALF to give an indication of induction of iNOS and inhalation of NO donors.

2. Materials and methods 2.1. Animals Groups of six male Dunkin – Hartley guinea-pigs, weighing 350 – 450 g were used throughout. Animals received food and water ad libitum and lighting was maintained in the room (22 ^ 2 8C) on a 12 h cycle. This work complied with the guidelines for the care and use of laboratory animals according to the Animals (Scientific Procedures) Act 1986. 2.2. Measurement of respiratory function Whole body plethysmography of the conscious guineapig was used to monitor airway function, recorded as specific airways conductance ðsGaw Þ: The method was as described by Griffiths-Johnson et al. [16], although a computerized data acquisition system replaced the original oscilloscope and angle resolver [17]. Animals were fitted with a face mask and placed in a restrainer which was then slid into the plethysmograph chamber. The computer ran AcqKnowledge software using a Biopack data acquisition system. This system was capable of acquiring and storing data referring to the air across a pneumotachograph (Mercury FIL) as the animal breathed. The resulting change in box volume (pressure) was simultaneously measured. Changes in air flow and box pressure were measured using UP1 and UP2 pressure transducers, respectively. The resulting waveforms could then be rapidly analysed by comparing the gradients of the flow and the box pressure waves at a point where flow tended towards zero, i.e. end tidal flow. A function of these two parameters, allowing for air pressure and the weight of the animal, gave the resulting value for specific airway conductance ðsGaw Þ. A minimum of five breaths were analysed for each animal at each time point. Before each experiment, the animals were handled and familiarized with the apparatus to reduce stress. Between recordings, animals were removed from the plethysmograph and placed in a holding cage. 2.3. Inhalation exposures 2.3.1. LPS exposures Animals were exposed for 1 h to a nebulized solution of LPS (30 mg ml21) or the LPS vehicle (0.9% pathogen-free saline) in a sealed chamber (620 £ 300 £ 420 mm). A Wright nebulizer, supplied with air at a pressure of 20 lb. p.s.i., was used to deliver the aerosol at a rate of 0.5 ml min21. sGaw was measured twice prior to exposure (baseline) and then at regular intervals (0, 15, 30, 45, 60 min) after the exposure. 2.3.2. Spasmogen exposures Airway reactivity to nebulized histamine (1 mM, 20 s) was assessed 24 h before and at 1 or 24 h after exposure to

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LPS or the LPS vehicle (normal saline), each time in a separate group of animals. This dose of spasmogen, delivered as a nose-only exposure, caused a small threshold bronchoconstriction. To examine airway hyporeactivity, or any residual bronchodilator effect at 24 h after exposure to LPS, a higher dose of histamine (nose-only, 3 mM, 20 s) was used, which produced a significant bronchoconstriction. Measurements of sGaw were taken before and at 0, 5 and 10 min after exposure to spasmogen. 2.3.3. Drug/vehicle exposures Other animals received molar equivalent concentrations (0.7 mM) of budesonide or NCX 1020; SNAP (1.4 mM) or SNAP plus budesonide (1.4, 0.7 mM, respectively) or vehicle (DMSO 30%, ethanol 30%, saline 40%) for 15 min at 24 h and 45 min before the LPS challenge. All drugs or vehicle were delivered as a whole body exposure (chamber: 300 £ 180 £ 180 mm). Measurements of sGaw were taken throughout the experiment and airway reactivity to histamine (nose-only, 1 mM, 20 s) was assessed 24 h before and at 1 and 24 h after LPS challenge. 2.4. Bronchoalveolar lavage fluid (BALF) Within 20 min of assessing airway reactivity to histamine, at 1 or 24 h after LPS or saline challenge, animals were overdosed with pentobarbitone sodium (400 mg kg21 i.p. Euthatalw) and the trachea cannulated. Normal saline (1 ml 100 g21, twice) was injected through the cannula into the lungs and recovered after 3 min. This was repeated and a total cell count (cells ml21) performed on the pooled sample using a Neubauer haemocytomer. 100 ml of the BALF fluid was then centrifuged using a Shandon Cytospin (Runcorn, Cheshire, UK) at 1000 rpm for 7 min. The resulting cytospin smear was differentially stained with Leishman’s stain (1.5% in 100% methanol, 6 min) and a minimum of 500 cells (macrophages, eosinophils and neutrophils) were counted. The remaining BALF sample was then centrifuged (1200 rpm, 6 min), the supernatant removed and frozen (2 20 8C). 2.4.1. Measurement of myeloperoxidase The levels of free myeloperoxidase (MPO) in the supernatant from the BALF were determined as an index of neutrophil activation according to a method described by Heuer et al. [18]. 100 ml samples (BALF or standards) were added to 100 ml of the reaction mixture consisting of 100 mM o-dianisidine dihydrochloride (indicator) and 0.1% Triton X, 100. After 30 min at 24 8C in the dark, the optical density at 450 nm was evaluated (MRX TC revelation Dynex technologies, Jencons-PLS, East Sussex, UK). When assaying BALF, 100 ml of supernatant was added to 100 ml of the reaction mixture containing 2 mM 3 amino-1,2,4-triazole (AMT, inhibitor of eosinophil peroxidase). Each sample was assayed in duplicate. For the standard curve, six known concentrations of horseradish

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peroxidase (0 – 200 mU ml21) in normal saline were added to the reaction mixture and a line drawn by linear regression from which the concentrations in BALF were determined. 2.4.2. Measurement of protein The levels of protein in the supernatant from the BALF were determined as an index of increased plasma exudation according to previously described methodology [19,20]. Coomassie blue reagent (100 mg) was dissolved in 50 ml 95% ethanol. To this solution, 100 ml 85% (w/v) phosphoric acid was added. The resulting solution was diluted to a final volume of 1 litre. Final concentrations in the reagent were 0.01% (w/v) Coomassie Brilliant Blue, 4.7% (w/v) ethanol, and 8.5% (w/v) phosphoric acid. This was left to stand for 24 – 48 h and then filtered to remove the natural precipitate that forms. 100 ml of six known concentrations of bovine serum albumin (0 –900 mg ml21) were added to 1 ml of Coomassie blue reagent. After 1 h at 24 8C, the optical density at 595 nm was evaluated (MRX TC revelation Dynex technologies, Jencons-PLS, East Sussex, UK) and a line drawn by linear regression. When assaying BALF, 30 ml of supernatant was added to 70 ml of normal saline together with 1 ml Coomassie blue reagent. Each sample was assayed in duplicate. 2.4.3. Measurement of NO metabolite levels (total nitrate and nitrite) The levels of the NO metabolite levels (total nitrate and nitrite) were determined as an index of NO production and inhalation of NO donors according to a method previously described [13]. One hundred microlitre of six known concentrations of sodium nitrate (0 – 20 mg ml21) in normal saline or 100 ml of supernatant from BALF were incubated (37 8C) for 30 min with HEPES buffer (50 mM, pH 7.4), FAD (5 mM), NADPH (0.1 mM), distilled water (290 ml) and nitrate reductase (0.2 U mL21) for nitrite conversion to nitrate. Any unreacted NADPH in the solution (500 ml) was then oxidised by incubating (25 8C) for 10 min with potassium ferricyanide (1 mM). The samples were then incubated (25 8C) with 1 ml of the Greiss reagent (NED: 0.2% (w/v), sulphanilamide: 2% (w/v) solubilized in double distilled water: 95% and phosphoric acid: 5% (v v21)), for 10 min. The plate was then read on a plate reader (MRX TC revelation Dynex technologies, Jencons-PLS, East Sussex, UK) at 543 nm to determine total nitrite (nitrate and reduced nitrite to nitrate) concentration. Each sample was assayed in duplicate. 2.5. Inhalation exposures against histamine-induced bronchoconstriction Further animals received a bronchoconstrictor dose of histamine (nose-only, 3 mM, 20 s). Fifteen minutes later they received inhaled NCX 1020 (0.7 mM) or vehicle (DMSO 30%, ethanol 30%, saline 40%) for 15 min

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and a further dose 24 h later. 2.5 h after the second exposure they were exposed to a second histamine challenge. This timing corresponds to that used for administration of NCX 1020 before LPS, allowing 1 h for the duration of LPS exposure and 1 h for the time after LPS before histamine challenge. Changes in % sGaw were measured 0, 5, 10 min after both histamine exposures. 2.6. Expression of results and statistical analysis Because of the intersubject variability of sGaw values, they were expressed as the percentage change from a baseline value of sGaw taken immediately prior to the start of a procedure. Differences in BAL fluid cell counts following LPS were compared with vehicle-treated LPS challenged animals using analysis of variance followed by Dunnett’s test. Immediate airway reactivity to histamine before and after LPS were compared using paired Student’s t-test. Differences were considered significant at P , 0:05:

2.7. Drugs and solutions Aspergillus nitrate reductase (NADPH:nitrate oxidoreductase) 2.0 U/ml was purchased from Boehringer Mannheim (Indianapolis, Indiana, USA). S-nitroso-N-acetylpenicillamine (SNAP) from Tocris (Northpoint Way, Avonmouth, UK). b-nicotinamide adenine dinucleotide phosphate reduced tetrasodium salt (NADPH), Coomassie blue reagent, dimethyl sulphoxide (DMSO), flavin adenine dinucleotide disodium salt dihydrate (FAD), histamine diphosphate salt, horseradish peroxidase (type 1), lipopolysaccharide (LPS: Escherchia coli O26:B6), N-(2hydroxyethyl)piperazine-N’-(2-ethanesulfonic acid-4-(2hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES) buffer, o-dianisidine dihydrochloride, phosphoric acid, potassium ferricyanide, sulphanilamide, Tris buffer (Tris[hydroxymethyl]aminomethane, Trizma) and 0.1% Triton X, 100 were purchased from Sigma (Poole, Dorset, UK.). Ethanol was purchased from Fisher (Loughborough, UK) Euthatalw (pentobarbital sodium 200 mg ml21) was

Fig. 1. (A) The responsiveness of the airways to a nose-only exposure to histamine (hist) (1 mM, 20 s) before and 1 h after exposure (60 min) to nebulized LPS in conscious guinea-pigs. Change in sGaw (baseline, 0, 5, 10 min after histamine) expressed as a percentage of the baseline sGaw values. (B) The immediate responsiveness of the airways to histamine (Hist) (1 mM, 20 s, nose-only) 24 h before and 1 h after; (1) LPS challenge in vehicle-treated animals; (2) saline challenge in untreated animals; LPS challenge in (3) untreated (4) budesonide, (5) NCX 1020 (NO-donating budesonide), (6) SNAP and (7) SNAP plus budesonide treated animals. Each point represents the mean ^ S.E.M. ðn ¼ 6Þ: Immediate change in sGaw expressed as a percentage of the baseline sGaw values. Negative values represent bronchoconstriction. Significance from baseline sGaw ð* p , 0:05Þ between immediate response to His 24 h before and 1 h after LPS challenge was determined by Student’s paired t-test.

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purchased from Rhone Merieux (Harlow, Essex, UK). Sterile saline (0.9% NaCl) was purchased from Baxter Healthcare, UK Budesonide and NCX 1020 were gifts from Nicox SA, Sophia Antipolis, France.

1.3 ^ 0.1a 0.1 ^ 0.04 1.2 ^ 0.1 ND 4.7 ^ 0.5a ND 1666.7 ^ 44.7a

b

Animals inhaled drug or vehicle 24 h and 45 mins before LPS. P , 0:05 (Dunnett’s test) when compared to vehicle-treated LPS challenged animals. P , 0:05 (paired t-test) when compared to histamine bronchoconstriction before LPS or saline challenge (AHR, airway hyperreactivity; ND, not detected; n.d., not determined).

3. Results

a

2.1 ^ 0.1a 0.07 ^ 0.02 1.1 ^ 0.1 0.9 ^ 0.03a 8.7 ^ 0.9a 83.0 ^ 8.4a n.d. 2.5 ^ 0.3 0.07 ^ 0.02 1.1 ^ 0.1 1.3 ^ 0.1 6.6 ^ 0.6 61.0 ^ 8.1a n.d. 1.9 ^ 0.1a 0.07 ^ 0.02 1.0 ^ 0.1 0.8 ^ 0.06a 12.0 ^ 2.5a 66.7 ^ 4.3a n.d. 2.3 ^ 0.3 0.07 ^ 0.03 1.1 ^ 0.1 1.2 ^ 0.2 5.6 ^ 1.9 123.6 ^ 18.7 n.d. 2.8 ^ 0.2 0.1 ^ 0.04 1.3 ^ 0.1 1.4 ^ 0.1 8.5 ^ 1.4 44.5 ^ 15.0a 2760 ^ 42a 1.4 ^ 0.1a 0.07 ^ 0.02 1.3 ^ 0.1 ND 18.3 ^ 2.3a ND 2000 ^ 40

224.0 ^ 5.3b AHR 220.2 ^ 1.8b AHR 26.4 ^ 3.1 26.5 ^ 2.0

220.3 ^ 3.8b AHR 21.7 ^ 3.5

0.2 ^ 4.3 23.0 ^ 2.7 25.2 ^ 3.5

22.1 ^ 3.1 25.6 ^ 4.1 22.4 ^ 4.6

Histamine 24 h before LPS or saline 21.2 ^ 2.2 challenge (% reduction in sGaw Þ Histamine 1 h after LPS or saline 223.9 ^ 4.6b AHR challenge (% reduction in sGaw Þ Total cells ml21 ( £ 106) 2.6 ^ 0.1 Eosinophils ml21 ( £ 106) 0.1 ^ 0.02 Macrophages ml21 ( £ 106) 1.25 ^ 0.1 Neutrophils ml21 ( £ 106) 1.3 ^ 0.1 Total nitrate and nitrite (mM) 4.2 ^ 1.6 Myeloperoxidase (MPO) (mU ml21) 122 ^ 14.9 Protein (mg ml21) 2120 ^ 40

SNAP þ budesonide Naive treatment þ LPS NCX 1020 SNAP treatment þ LPS treatment þ LPS Budesonide treatment þ LPS LPS Vehicle-treatment þ LPS Saline

Table 1 Immediate % reduction in specific airways conductance ðsGaw Þ, histamine (1 mM) reactivity 24 h before and 1 h after lipopolysaccharide (LPS) or saline challenge and total leukocyte counts, differential macrophage, neutrophil and eosinophil counts and total nitrate and nitrite, neutrophil myeloperoxidase (MPO) and protein levels in BAL samples 1 h after LPS or saline challenge

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3.1. Effects of exposure to LPS or saline 3.1.1. 1 h after LPS or saline Exposure to nebulised saline or LPS caused no change in % sGaw for 1 h after challenge. Inhalation of histamine (1 mM, 20 s) caused a threshold bronchoconstriction 24 h before LPS (5.6 ^ 4.1% reduction in sGaw Þ but at 1 h after low-dose LPS challenge it caused a significantly increased bronchoconstriction (23.9 ^ 5.3 reduction in sGaw Þ indicating AHR (Fig. 1, Table 1). There was a substantial increase in total number of cells in BALF 1 h after LPS when compared to saline challenged animals (2.8 ^ 0.2, 1.4 ^ .0.1 £ 106 ml21, respectively) (Fig. 2, Table 1). There was neutrophil (1.4 ^ 0.1 £ 106 ml21) influx 1 h after LPS compared to no neutrophil influx in saline challenged animals (Fig. 2, Table 1). There was, however, no change in macrophage or eosinophil numbers in BALF 1 h after LPS when compared to saline challenged animals (Fig. 2, Table 1). There were similar levels of total cell, macrophage and eosinophil numbers in BALF of naı¨ve animals when compared to saline challenged animals. No neutrophils were detected in BALF of naı¨ve animals. MPO was detected in BALF 1 h after LPS challenge (44.5 ^ 9.0 mU ml21) but not 1 h after saline challenge (Fig. 3, Table 1). There was a substantial increase in protein (2760 ^ 42, 2100 ^ 40 mg ml21, respectively) (Table 1) but a reduction in NO metabolite (8.5 ^ 1.4, 18.3 ^ 2.3 mM, respectively) (Fig. 4, Table 1) levels in BALF 1 h after LPS challenge compared with saline challenge. There were substantially less NO metabolite (4.7 ^ 0.5 mg ml21) and protein (1666.7 ^ 44.7 mg ml21) levels in naı¨ve animals when compared with saline challenged animals. 3.1.2. 24 h after LPS or saline Inhalation of histamine (1 mM, 20 s) prior to and 24 h after saline (2 2.2 ^ 2.3, 2 0.01 ^ 2.5% reductions in sGaw , respectively) or LPS (2 1.6 ^ 1.7, 2 2.5 ^ 1.4% reductions in sGaw , respectively) failed to produce a significant bronchoconstriction (Table 2). Exposing the guinea-pigs to a higher dose of inhaled histamine (3 mM, 20 s) before LPS exposure caused a significant bronchoconstriction (2 25.3 ^ 7.2, reduction in sGaw , respectively). The same dose, 24 h after LPS produced a similar bronchoconstriction (2 22.0 ^ .3.5% reduction in sGaw Þ (Fig. 5). Thus, there was no hyper- or hypo-reactivity. There was a substantial increase in the total number of cells (8.9 ^ 0.7 £ 106 ml21), neutrophils (5.5 ^ 0.3 £ 106 ml21),

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Fig. 2. Differential cell counts in bronchoalveolar lavage fluid (BALF) removed from guinea-pigs 1 h after, nebulised (60 min) LPS with inhaled vehicle (DMSO 30%, ethanol 30%, saline 40%, 15 min); nebulised saline challenge only; nebulised LPS challenge only; nebulised LPS with budesonide (0.7 mM, 15 min), NCX 1020 (1.4 mM, 15 min), SNAP (1.4 mM, 15 min), SNAP (1.4 mM, 15 min) with budesonide (0.7 mM, 15 min); or naı¨ve animals. Treatment was administered by inhalation (15 min) 24 h and 45 min before LPS challenge. Each point represents the mean ^ SEM. ðn ¼ 6Þ of the cells per sample ( £ 106). Significant differences in the cells ð* P , 0:05Þ retrieved were compared with cells from vehicle-treated, LPS challenged animals and compared with ANOVA, followed by Dunnett’s post hoc analysis.

macrophages (3.1 ^ 0.4 £ 106 ml 21) and eosinophils (0.4 ^ 0.07 £ 106 ml21) in BALF 24 h after LPS when compared to saline challenged animals (1.1 ^ 0.1, 0.007 ^ 0.006, 1.1 ^ 0.1, 0.03 ^ .0.007 £ 106 ml 21 ,

respectively) (Fig. 6, Table 2). There were similar levels of total cell, macrophage and eosinophil number in BALF of naı¨ve animals when compared to saline challenged animals. No neutrophils were detected in BALF of naı¨ve animals.

Fig. 3. Neutrophil myeloperoxidase (MPO) in bronchoalveolar lavage fluid (BALF) removed from guinea-pigs (A) 1 h and (B) 24 h after, nebulised (60 min) LPS (30 mcg ml21) with inhaled vehicle (DMSO 30%, ethanol 30%, saline 40%, 15 min); nebulised saline challenge only; nebulised LPS challenge only; nebulised LPS with budesonide (0.7 mM, 15 min), NCX 1020 (1.4 mM, 15 min), SNAP (1.4 mM, 15 min), SNAP (1.4 mM, 15 min) with budesonide (0.7 mM, 15 min); or naı¨ve animals. Treatment was administered by inhalation (15 min) 24 h and 45 min before LPS challenge. Each point represents the mean ^ S.E.M. ðn ¼ 6Þ of MPO concentration per sample (mU ml21). Significant differences in MPO concentrations ð* P , 0:05Þ were compared with concentrations from vehicle-treated, LPS challenged animals and compared with ANOVA, followed by Dunnett’s post hoc analysis.

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2 Fig. 4. Total nitrite (NO2 2 ) and nitrate (NO3 ) in bronchoalveolar lavage fluid (BALF) removed from guinea-pigs (A) 1 h and (B) 24 h after, nebulised (60 min) LPS with inhaled vehicle (DMSO 30%, ethanol 30%, saline 40%, 15 min); nebulised saline challenge only; nebulised LPS challenge only; nebulised LPS with budesonide (0.7 mM., 15 min), NCX 1020 (1.4 mM, 15 min), SNAP (1.4 mM, 15 min), SNAP (1.4 mM, 15 min) with budesonide (0.7 mM, 15 min); or naı¨ve animals. Treatment was administered by inhalation (15 min) 24 h and 45 min before LPS challenge. Each point represents the mean ^ S.E.M. ðn ¼ 6Þ of the 2 2 2 2 total NO2 3 and NO2 concentration per sample (mM). Significant differences in total NO3 and NO2 concentrations ð* P , 0:05Þ were compared with total NO3 2 and NO2 concentrations (mM) from vehicle-treated, LPS challenged animals and compared with ANOVA, followed by Dunnett’s post hoc analysis.

There was a substantial increase in MPO (68.7 ^ 7.8, 21.3 ^ 13.5 mU ml21, respectively) (Fig. 3, Table 2) and protein (2100 ^ 40, 1870 ^ 50 mg ml21, respectively) (Fig. 7, Table 2) levels in BALF 24 h after LPS challenge compared with 24 h after saline challenge. NO metabolite levels were increased in LPS challenged animals compared to saline challenged (15.2 ^ 2.5, 8.0 ^ 2.6 mM, respectively) (Fig. 4, Table 2). There were substantially less NO metabolite (4.7 ^ 0.5 mg ml21) (Fig. 4, Table 2) and protein (1666.7 ^ 44.7 mg ml21) (Fig. 7, Table 2) levels in naı¨ve animals when compared with saline challenged animals. 3.2. Effects of vehicle on LPS responses 3.2.1. 1 h after LPS Vehicle pretreatment did not affect the AHR to histamine (1 mM, 20 s) 1 h after LPS compared to before LPS (2 1.2 ^ 2.2, 2 23.9 ^ 4.6% reductions in sGaw Þ (Fig. 1, Table 1). Vehicle did not change cells numbers in BALF 1 h after LPS when compared to untreated LPS challenged animals (Fig. 2, Table 1). There were significantly less NO metabolite (4.2 ^ 1.6 mM) (Fig. 4, Table 1) levels and significantly more MPO (122.0 ^ 14.9 mU ml21) (Fig. 3, Table 1) in BALF 1 h after LPS in vehicle-treated animals when compared to untreated LPS challenged animals. Protein levels in vehicle-treated animals (2120 ^ 40 mg ml21)

approached levels seen in saline challenged animals (Table 1). It was not therefore appropriate to measure protein levels at 1 h after LPS in subsequent studies using drugs prepared in this vehicle. 3.2.2. 24 h after LPS At 24 h after LPS, there was no bronchoconstriction to histamine (1 mM, 20 s) before or 24 h after LPS (2 0.1 ^ 1.6, 2 1.0 ^ 1.7% reductions in sGaw , respectively) (Table 2). Compared with untreated, in LPS challenged animals there was similar total cell (9.5 ^ 0.4 £ 106 ml21), neutrophil (6.0 ^ 0.5 £ 106 ml21), macrophage (3.2 ^ 0.3 £ 106 ml21) and eosinophil numbers (0.4 ^ 0.09 £ 106 21) in BALF, 24 h after LPS (Fig. 6, Table 2). There was significantly more protein (2480 ^ 70 mg ml21) (Fig. 7, Table 2) in BALF 24 h after LPS in vehicle-treated animals compared with untreated LPS challenged animals. There were similar levels of MPO (Fig. 3, Table 2) and NO metabolites (Fig. 4, Table 2) in BALF 24 h after LPS in vehicle-treated animals compared with untreated LPS challenged animals. The bronchoconstriction to the larger bronchoconstricting dose of histamine (3 mM) was also not affected by administrations of vehicle (2 28.3 ^ 2.7, 2 24.3 ^ 2.2% reductions in sGaw , respectively) 26 and 2.5 h before the second histamine exposure (Fig. 5). These animals did not receive either saline or LPS exposures.

7.4 ^ 0.6a 0.2 ^ 0.05a 3.8 ^ 0.3 3.4 ^ 0.4a 8.8 ^ 1.7 ND 2730 ^ 110 10.3 ^ 0.7 0.7 ^ 0.2 4.5 ^ 0.4a 5.2 ^ 0.4 10.6 ^ 1.4 74.3 ^ 12.9 2660 ^ 200 5.6 ^ 0.4a 0.1 ^ 0.05a 3.1 ^ 0.4 2.4 ^ 0.2a 7.2 ^ 0.6a ND 2390 ^ 80 10.2 ^ 1.1 0.06 ^ 0.03a 3.7 ^ 0.3 6.4 ^ 0.8 5.5 ^ 1.6a 70.3 ^ 15.0 2690 ^ 330a 8.9 ^ 0.7 0.4 ^ 0.07 3.1 ^ 0.4 5.5 ^ 0.3 15.2 ^ 2.5 68.7 ^ 7.8 2100 ^ 40a 1.1 ^ 0.1a 0.03 ^ 0.007a 1.1 ^ 0.1a 0.007 ^ 0.006a 8.0 ^ 2.6a 21.3 ^ 13.5a 1870 ^ 50a 9.5 ^ 0.4 0.4 ^ 0.09 3.2 ^ 0.3 6.0 ^ 0.5 15.2 ^ 3.6 72.0 ^ 10.0 2480 ^ 70

3.3.1. 1 h after LPS There was a significant bronchoconstriction to histamine (1 mM, 20 s) 1 h after LPS compared to before LPS (2 5.2 ^ 3.5, 2 20.2 ^ 1.8% reductions in sGaw , respectively) (Fig. 1, Table 1) indicating AHR. Budesonide did not change cell numbers in BALF 1 h after LPS compared with vehicle-treated animals (Fig. 2, Table 1). There was no change in MPO (Fig. 3, Table 1) and NO metabolite (Fig. 4, Table 1) levels when compared to vehicle-treated LPS challenged animals.

P , 0:05 (Dunnett’s test) when compared to vehicle-treated LPS challenged animals. (ND, not detected).

0.2 ^ 1.8 21.5 ^ 2.4 20.9 ^ 3.5 2.5 ^ 2.6 21.6 ^ 1.1 20.01 ^ 2.5 21.0 ^ 1.7

3.3. Effects of budesonide

a

1.4 ^ 2.6 0.2 ^ 2.1 22.5 ^ 1.3 20.5 ^ 2.6 22.5 ^ 1.4 22.2 ^ 2.3 20.1 ^ 1.6

Histamine 24 h before LPS or saline challenge (% reduction in sGaw Þ Histamine 24 h after LPS or saline challenge (% reduction in sGaw Þ Total cells ml21 ( £ 106) Eosinophils ml21 ( £ 106) Macrophages ml21 ( £ 106) Neutrophils ml21 ( £ 106) Total nitrate and nitrite (mM) Myeloperoxidase (MPO) (mU ml21) Protein (mg ml21)

Saline

LPS

SNAP treatment þ LPS NCX 1020 treatment þ LPS Budesonide treatment þ LPS

SNAP þ budesonide treatment þ LPS

Naive

1.3 ^ 0.1a 0.1 ^ 0.04 1.2 ^ 0.1 ND 4.7 ^ 0.5a ND 1666.7 ^ 44.7a

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Vehicle-treatment þ LPS

Table 2 Immediate % reduction in specific airways conductance ðsGaw Þ, histamine (1 mM) reactivity 24 h before and 24 h after lipopolysaccharide (LPS) or saline challenge and total leukocyte counts, differential macrophage, neutrophil and eosinophil counts and total nitrate and nitrite, neutrophil myeloperoxidase (MPO) and protein levels in BALF samples 24 h after LPS or saline challenge. Animals inhaled drug or vehicle 24 h and 45 mins before LPS challenge

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3.3.2. 24 h after LPS There was no bronchoconstriction to histamine (1 mM, 20 s) 24 h after LPS (Table 2) in budesonide treated animals. Budesonide significantly reduced eosinophil influx (0.06 ^ 0.03 £ 106 ml21) (Fig. 6, Table 2) compared to vehicle but did not affect total cell, macrophage and neutrophil numbers in BALF 24 h after LPS (Fig. 6, Table 2). There was no change in MPO (Fig. 3, Table 2) and protein (Fig. 7, Table 2) in budesonide-treated animals, 24 h after LPS when compared to vehicle-treated animals. There was a significant reduction in NO metabolite levels (5.5 ^ 1.6 mM) (Fig. 4, Table 2) in budesonide-treated LPS challenged animals when compared to vehicle-treated LPS challenged animals. 3.4. Effects of NCX 1020 3.4.1. 1 h after LPS NCX 1020 blocked AHR to histamine at 1 h after LPS challenge, the reductions in sGaw before and after LPS were identical (2 2.1 ^ 3.1, 2 6.4 ^ 3.1% reductions in sGaw , respectively) (Fig. 1, Table 1) and significantly reduced total cell (1.9 ^ 0.1 £ 106 ml21) and neutrophil (0.8 ^ 0.06 £ 106 ml21) influx (Fig. 2, Table 1) 1 h after LPS when compared to vehicle-treated animals. There was a significant reduction in MPO (66.7 ^ 4.3 mU ml21) (Fig. 3, Table 1) levels and a significant increase in NO metabolite levels (12.0 ^ 2.5 mM) (Fig. 4, Table 1) in BALF 1 h after LPS in NCX 1020-treated animals when compared to vehicle-treated animals. 3.4.2. 24 h after LPS There was no bronchoconstriction to histamine (1 mM, 20 s) 24 h after LPS in NCX 1020 treated animals (Table 1). NCX 1020 significantly reduced total cell (5.6 ^ 0.4 £ 106 ml21), neutrophil (2.4 ^ 0.2 £ 106 ml21) and eosinophil (0.1 ^ 0.05 £ 106 ml21) numbers (Fig. 6, Table 2) when compared to vehicle but did not affect macrophage numbers (3.1 ^ 0.4 £ 106 ml21) (Fig. 6, Table 2) in BALF, 24 h after LPS. MPO was not detected in BALF 24 h after LPS in NCX 1020-treated animals (Fig. 3, Table 2). There was a significant reduction in NO metabolite levels (7.2 ^ 0.6 mM) (Fig. 4, Table 2) 24 h after LPS in NCX

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1020-treated animals when compared to vehicletreated animals. There was no change in protein levels (Fig. 7, Table 2) when compared to vehicle-treated animals. The higher bronchoconstricting dose of histamine (3 mM) produced a similar bronchoconstriction after NCX 1020 (administered 26 and 2.5 h beforehand) as 30 min before the first exposure to NCX 1020 (2 24.2 ^ 3.7, 2 28.1 ^ 4.0% reductions in sGaw , respectively) (Fig. 5). 3.5. Effects of SNAP 3.5.1. 1 h after LPS In SNAP-treated animals, there was AHR to histamine (1 mM, 20 s) 1 h after LPS, the response after LPS was significantly greater than before (2 20.3 ^ 3.8, 2 3.0 ^ 2.7% reductions in sGaw , respectively) (Fig. 1, Table 1). SNAP did not change cell numbers in BALF 1 h after LPS compared with vehicle-treated animals (Fig. 2, Table 1). There was a significant reduction in MPO (61.0 ^ 8.1 mU ml21) (Fig. 3, Table 1) levels in BALF 1 h after LPS in SNAP-treated animals when compared to vehicle-treated animals. There was no change in NO metabolite levels (6.6 ^ 0.6 mM) (Fig. 4, Table 1) when compared to vehicle-treated animals. 3.5.2. 24 h after LPS There was no bronchoconstriction to histamine (1 mM, 20 s) 24 h after LPS in SNAP-treated animals (Table 1). SNAP significantly increased macrophage numbers (4.5 ^ 0.4 £ 106 ml21) when compared to vehicle but did not affect total cell, neutrophil or eosinophil numbers in BALF, 24 h after LPS (Fig. 6, Table 2). There was no change in MPO (Fig. 3, Table 2) or protein (Fig. 7, Table 2) levels in BALF, 24 h after LPS in SNAP treated animals. NO metabolites were reduced by SNAP but did not reach significance (10.6 ^ 1.4 mM, P ¼ 0:097Þ (Fig. 4, Table 2). 3.6. Effects of SNAP plus budesonide

Fig. 5. The responsiveness of the airways to a nose-only exposure to histamine (Hist) (3 mM, 20 s) (a) 24 h before and 24 h after nebulised LPS exposure (60 min); (b) 24 h before and 2.5 h after final exposure (15 min) to nebulised vehicle (30% DMSO, 30% ethanol, 40% saline); (c) 24 h before and 2.5 h after final exposure (15 min) to nebulised NCX 1020 (0.7 mM,). Vehicle or NCX 1020 was administered by inhalation (15 min) 26 h (30 min following first histamine exposure) and 2.5 h (26.5 h after first histamine exposure) before second Hist exposure. Each point represents the mean ^ S.E.M. ðn ¼ 6Þ: Change in sGaw (baseline, 0, 5, 10 min after histamine) expressed as a percentage of the baseline sGaw values. Negative values represent bronchoconstriction. Immediate response before and after exposure, determined by Student’s paired t-test was not significant ðP > 0:05Þ.

3.6.1. 1 h after LPS Like NCX 1020, SNAP plus budesonide blocked AHR to histamine at 1 h after LPS (0.2 ^ 4.3, 2 1.7 ^ 3.5% changes in sGaw , before and after LPS, respectively) (Fig. 1, Table 1). Total cell (2.1 ^ 0.1 £ 106 ml21) and neutrophil influx (0.9 ^ 0.03 £ 106 ml21) were significantly reduced 1 h after LPS when compared to vehicletreated animals (Fig. 2, Table 1). There was a significant reduction in MPO (83.0 ^ 8.4 mU ml21) (Fig. 3, Table 1) levels and increased NO metabolite levels (8.7 ^ 0.9 mM) (Fig. 4, Table 1) in BALF 1 h after LPS in SNAP plus budesonide when compared to vehicle-treated animals. 3.6.2. 24 h after LPS There was no bronchoconstriction to histamine (1 mM, 20 s) 24 h after LPS in SNAP plus budesonide treated

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Fig. 6. Differential cell counts in bronchoalveolar lavage fluid (BALF) removed from guinea-pigs 24 h after, nebulised (60 min) LPS with inhaled vehicle (DMSO 30%, ethanol 30%, saline 40%, 15 min); nebulised saline challenge only; nebulised LPS challenge only; nebulised LPS with budesonide (0.7 mM, 15 min), NCX 1020 (1.4 mM, 15 min), SNAP (1.4 mM, 15 min), SNAP (1.4 mM, 15 min) with budesonide (0.7 mM, 15 min); or naı¨ve animals. Treatment was administered by inhalation (15 min) 24 h and 45 min before LPS challenge. Each point represents the mean ^ S.E.M. ðn ¼ 6Þ of the cells per sample ( £ 106). Significant differences in the cells ð* P , 0:05Þ retrieved were compared with cells from vehicle-treated, LPS challenged animals with ANOVA, followed by Dunnett’s post hoc analysis.

animals (Table 1) Like NCX 1020, SNAP plus budesonide reduced total cell (7.4 ^ 0.6 £ 106 ml21), neutrophil (3.4 ^ 0.4 £ 106 ml 21) and eosinophil (0.2 ^ 0.05 £ 106 ml 21) numbers but did not affect

macrophage numbers (3.8 ^ 0.3 £ 106 ml21) in BALF, 24 h after LPS when compared to vehicle (Fig. 6, Table 2). MPO (Fig. 3, Table 2) was not detected in BALF 24 h after LPS in SNAP plus budesonide-treated animals.

Fig. 7. Protein in bronchoalveolar lavage fluid (BALF) removed from guinea-pigs 24 h after, nebulised (60 min) LPS with inhaled vehicle (DMSO 30%, ethanol 30%, saline 40%, 15 min); nebulised saline challenge only; nebulised LPS challenge only; nebulised LPS with budesonide (0.7 mM, 15 min), NCX 1020 (1.4 mM, 15 min), SNAP (1.4 mM, 15 min), SNAP (1.4 mM, 15 min) with budesonide (0.7 mM, 15 min); or naı¨ve animals. Treatment was administered by inhalation (15 min) 24 h and 45 min before LPS challenge. Each point represents the mean ^ S.E.M. ðn ¼ 6Þ of the protein concentration per sample (mg ml21). Significant differences in protein concentrations ð* P , 0:05Þ were compared with protein concentrations from vehicle-treated, LPS challenged animals with ANOVA, followed by Dunnett’s post hoc analysis.

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There was no change in protein (Fig. 7, Table 2) levels when compared to vehicle-treated animals. NO metabolites were reduced by SNAP plus budesonide but did not reach significance (8.8 ^ 1.7 mM, P ¼ 0:088Þ (Fig. 4, Table 2).

4. Discussion As previously reported by Toward and Broadley [13], there was increased AHR to histamine associated with increased neutrophil influx and reduced NO metabolite levels, 1 h after LPS when compared to saline challenged animals. As reported by Toward and Broadley [13] there was also increased NO metabolite levels in BALF 1 and 24 h following saline challenge when compared to naı¨ve animals possibly due to the shear stress of inhalation of vehicle. As reported by Toward and Broadley [13] there was an increase in BALF neutrophil numbers 1 and 24 h following LPS compared to saline challenge. In the present study, administration of the NO derivative of budesonide, NCX 1020, or co-administration of a molar equivalent dose of budesonide with the NO donor, SNAP, reduced neutrophilia and AHR 1 h after LPS and neutrophilia 24 h after LPS. This was not observed following administration of SNAP or budesonide alone indicating that NCX 1020 is more potent than equimolar budesonide in reducing AHR and neutrophilia in this model of LPS-induced neutrophilia. A significant reduction in BALF NO metabolite levels was noted 1 h after LPS in untreated LPS challenged animals when compared to saline challenged animals. Vehicle treatment was found to potentate this reduction. This is likely to be due to the DMSO component of the vehicle which, having free radical scavenger properties [21], may reduce the metabolism of NO to the assayed metabolites, nitrate and nitrite. NCX 1020 and budesonide plus SNAP increased NO metabolite levels when compared to vehicle 1 h after LPS. The metabolism of the NO component of these drugs may simply have increased NO metabolite levels. This was not, however, noted with SNAP administration, 1 h after LPS. The vehicle may have interfered with the metabolism of the NO donors. The anti-inflammatory properties of NCX 1020 and budesonide plus SNAP may have prevented the deficiency of NO from occurring 1 h after LPS. NCX 1020 and SNAP plus budesonide blocked AHR 1 h after LPS challenge. We have shown that this dose of NCX 1020 is bronchodilatory against histamine when given 15 min before histamine [22]. It may be speculated that the NO component of these drugs by being administered 26 and 2.5 h before histamine simply exerted a bronchodilatory action 1 h after LPS thereby blocking AHR. When, however, NCX 1020 was given 26 h and 2.5 h before a bronchoconstrictive dose of histamine, bronchoconstriction to histamine did not change compared to histamine before drug administration. This indicates that the bronchodilatory effects of NCX 1020 had worn

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off by the time histamine was given after LPS. Since NO exerts an inhibitory modulatory effect on airway tone, it may be speculated that the deficiency of NO 1 h after LPS contributes to AHR. NCX 1020 and budesonide plus SNAP appear to restore NO levels 1 h after LPS and compensate for this deficiency. Since NCX 1020 and budesonide plus SNAP also reduced neutrophilia 1 h after LPS, it may be speculated that AHR may be caused by neutrophil-associated cytotoxicity [23]. NCX 1020 or budesonide plus SNAP, unlike SNAP alone did reduce neutrophilia 1 and 24 h after LPS. SNAP did, however, reduce the MPO in BALF 1 h after LPS suggesting that SNAP reduced neutrophil activation by a mechanism independent of a reduction in neutrophil numbers. The NO component of NCX 1020 and budesonide plus SNAP may, like SNAP, have had a direct inhibitory effect on neutrophil activation as evidenced by the fall in MPO levels. Exogenously administered NO has been shown to reduce neutrophil production of reactive oxygen species [24]. The direct inhibitory effect of SNAP on neutrophil activation was worn off, however, 24 h after LPS. In NCX 1020- and budesonide plus SNAP-treated animals, MPO levels in BALF, 24 h after LPS were not detected, possibly due to the reduction in neutrophil influx rather than their inactivation. Budesonide did not affect total cell, neutrophil and macrophage numbers in BALF, 1 or 24 h following LPS challenge. This is consistent with the known resistance of the guinea-pigs to corticosteroids compared with humans [25]. Similarly, prednisolone while reducing vascular permeability and AHR did not reduce total cell numbers in BALF, 2 h following LPS, in the guinea-pig [26]. In this present study, however, budesonide did reduce eosinophil influx, 24 h after LPS. Whelan et al. [25] similarly demonstrated that the corticosteroid, dexamethasone (i.p.) preferentially reduced platelet-activating factorinduced eosinophilia over LPS-induced neutrophilia in the guinea-pig lung. It has been suggested that COPD patients with eosinophilia may more likely respond to corticosteroid administration than those with little or no eosinophil influx [4]. Rather than being due to a reduction in neutrophil influx, this subset of COPD patients may respond to corticosteroid administration due to a reduction in eosinophilia-associated cytotoxicity. The neutrophil appears, however, to be the predominant and most cytotoxic inflammatory cell in COPD [2]. Superoxide [27], MPO [25] and neutrophil elastase [2] are produced by the neutrophil and may play a role in the pathophysiology of COPD. Neutrophil elastase may be the most cytotoxic agent causing mucus cell hyperplasia [28], epithelial damage, AHR and bronchoconstriction in the guinea-pig [23]. Although budesonide did not reduce total cell and neutrophil numbers, an equimolar dose of NCX 1020 or budesonide plus SNAP was effective. It may be speculated that the NO component of NCX 1020 by being

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bronchodilatory increased the inhaled dose of budesonide causing the reduction in neutrophil numbers and AHR. Fiorucci et al. [29] recently showed that subcutaneous administration of the NO-donating derivative of prednisolone (NCX 1015) further reduced colon neutrophil MPO levels in 2, 4, 6-trinitrobenzene-induced colitis in mice when compared to prednisolone administration. This suggests that co-administration of NO and corticosteroid by a non-inhalation route also gives a greater anti-inflammatory profile than corticosteroid administration alone. There were increased protein levels in BALF, 1 and 24 h following saline challenge when compared to naı¨ve animals. The shear stress of inhalation of saline vehicle may have increased plasma exudation. There was a substantial increase in protein levels in BALF 24 h following LPS challenge in untreated animals when compared to saline challenge. Vehicle, however, substantially reduced increased protein levels in BALF, 1 h after LPS towards saline challenged levels. This effect had worn off by 24 h where vehicle treatment further increased BALF protein levels 24 h following LPS challenge. In Yorkshire swine, inhaled NO for 4.5 h significantly reduced neutrophil and protein levels in BALF and MPO in lung tissue 5 h following Pseudmononas aeruginosa-induced sepsis [30]. Similarly, inhaled NO for 6 h attenuated neutrophil numbers in BALF 6 h following intravenous administration of LPS in the rabbit [31]. NCX 1020 while reducing neutrophil and MPO levels did not, however, reduce protein levels, 24 h following LPS challenge. There were increased NO metabolite levels 24 h after LPS in untreated, LPS challenged animals compared with saline challenged animals indicating the induction of iNOS 24 h after LPS. iNOS is induced by LPS which by forming excess NO may contribute to AHR, oedema and inflammation [6]. Administration of NO to a system where iNOS is already induced such as in COPD or in our model would be expected to further increase peroxynitrite formation by combination of NO with superoxide and thus nitrotyrosine and its associated deleterious effects. SNAP did not, however, cause a reduction in specific airways conductance, increase airway reactivity to a threshold dose of histamine or increase neutrophilia or eosinophilia 24 h after LPS. SNAP administration did, however, increase macrophage numbers when compared to vehicle-treated animals. This was possibly due to increased inhalation of LPS by bronchodilatory SNAP rather than due to a pro-inflammatory effect of SNAP per se. Spruntulis and Broadley [32] previously reported increased total cell, macrophage and eosinophil numbers in salbutamol-treated guinea-pigs, 24 h following OA challenge when compared to untreated OA challenged guinea-pigs. The anti-inflammatory effects of NCX 1020 may have counteracted the effects of the increased LPS inhalation. The corticosteroid, budesonide, by inhibiting iNOS [33] may remove excess, deleterious levels of iNOS-derived NO. This would explain the reduction in NO metabolite levels

24 h after LPS which were reduced in budesonide- and NCX 1020-treated animals. SNAP plus budesonide also substantially reduced NO metabolite levels but not to a significant level ðp ¼ 0:088Þ: Budesonide by inhibiting iNOS would offset any increase in NO metabolite levels resulting from the metabolism of SNAP or NCX 1020. SNAP did not, however, increase NO metabolite levels 24 h after LPS challenge. As previously discussed, this may be due to the free radical properties of the vehicle. The vehicle did not, however, reduce the increase in BALF NO metabolite levels arising from the induction of iNOS, 24 h after LPS challenge. As previously discussed, vehicle further reduced NO metabolite levels, 1 h after LPS suggesting that this effect had worn off, 24 h following LPS challenge. It is possible that the NO released from SNAP or NCX 1020 is metabolised to nitrate and nitrite shortly after LPS challenge and that these metabolites have been excreted, 24 h later [34]. iNOS-derived NO by being released 24 h after LPS challenge persists, giving increased NO metabolite levels, 24 h after LPS challenge. SNAP, like budesonide, however reduced NO metabolites, 24 h following LPS but not to significant levels ðp ¼ 0:097Þ: SNAP may decrease NO metabolites via a negative feedback system on iNOSderived NO production [35]. SNAP by inhibiting the production of inflammatory mediators which cause the induction of iNOS [6,36] may reduce iNOS-derived NO, 24 h following LPS. Budesonide and NO, by inhibiting iNOS and the production of mediators that cause its induction may reduce the increase in iNOS-derived NO, 24 h following LPS challenge. Inhibition of iNOS by corticosteroid is thought to occur at transcriptional level and may involve inhibition of activated transcription factor nuclear factor-kB (NF-kB) [33]. NF-kB also regulates the expression of the proinflammatory cytokines, IL-1b and TNF-a; chemokines, MIP-1a and MCP-1 which attract inflammatory cells into sites of inflammation; adhesion molecules such as VCAM-1 and ICAM-1 which are involved in the adhesion of inflammatory cells in the airways and inflammatory receptors (neurokinin-1, neurokinin-2, bradykinin-1 and IL-2) [37]. Glucocorticosteroids also inhibit activator protein-1 (AP-1) which regulates the expression of cytokine, IL-5 which chemo-attracts and prolongs the life of the eosinophil [33]. This additional effect on IL-5 may explain why budesonide was more effective in reducing eosinophilia than neutrophilia in BALF, 24 h after LPS. Corticosteroids may also reduce eosinophilia by increasing the apoptosis of eosinophils [33]. NO inhaled for 16 h was also found to reduce epithelial nitrotyrosine staining in lung tissue 16 h following LPS challenge in the rat possibly via a negative feedback system on cNOS and iNOS-derived NO production or by reducing superoxide [35]. Administration of NO may also shift the equilibrium towards reduced peroxynitrite and nitrotyrosine formation which would attenuate their associated deleterious effects [38]. High levels of local iNOS-derived NO may

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be deleterious while cNOS-derived NO, formed in smaller amounts may not exert toxic effects. Administration of a NO donor may be distributed throughout the lung and not localised to a specific superoxide-rich area to exert a deleterious effect. Honda et al. [35] reported reduced neutrophil numbers in BALF and myeloperoxidase in lung tissue, 16 h following inhaled NO in LPS challenged rats. NO by mopping up superoxide reduces hydrogen peroxide formation resulting in reduced adhesion and sequestration of neutrophils into the lung. Furthermore, by generating cGMP, NO also reduces chemotaxis [8]. In ischaemia/ reperfusion injury of distal microvascular beds in the cat, inhaled NO was shown to reduce oxidant-dependent leukocyte rolling, adhesion and emigration and endothelial dysfunction [9]. NF-kB activation in rats has been shown to mediate the neutrophil accumulation in BALF, 2 h after LPS challenge [39]. LPS administered to the mouse results in increased cytokines, TNF-a and IL-6 in lung homogenates 6 h later. This increase may be potentiated by the NOS inhibitor L-NAME. When murine macrophages, being the main sources for increases in TNF-a, were exposed to LPS, NF-kB binding increased which was further potentiated by L-NAME but attenuated by the NO donor, sodium nitroprusside, suggesting that NO may reduce the LPS-induced increases in the pro-inflammatory transcription factor, NF-kB and its associated pro-inflammatory mediators. L-NAME did not, however, further increase neutrophils or macrophages on lung sections following LPS [36]. Exogenous NO has also been shown to suppress the adhesion molecules, VCAM-1 and ICAM-l possibly via a reduction in NF-kB binding activity [40,41]. Inhaled NO administered for 6 h was found to inhibit LPS-induced increases in BALF protein, lactate dehydrogenase, reactive oxygen species, IL-8 in macrophages and neutrophilia associated with reduced NF-kB activation in macrophages [31]. SNAP or budesonide did not reduce neutrophil numbers 1 and 24 h after LPS. Administration of NCX 1020 or co-administration of SNAP and budesonide may have synergistically or additively reduced transcription factors and inflammation. By co-administering budesonide with SNAP or administering NCX 1020, transcription factor activation may have been sufficiently blocked to cause a reduction in neutrophilia and associated AHR. The dose 24 h before LPS is likely to have primed transcription factors, a further dose 45 min before LPS would reenforce this effect. Corticosteroids may also have direct inhibitory effects on cell function and chemotaxis [33]. The increased cGMP formation and ‘mopping up’ of superoxide by NO together with other anti-inflammatory effects by budesonide, including the inhibition of iNOS, may also have contributed to this synergistic or additive effect. Unlike in asthma, corticosteroids have been shown to be ineffective in reducing neutrophilia in COPD [4]. In our study, budesonide did not reduce neutrophilia in this

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LPS model. In our model, Toward and Broadley [42] previously showed a reduction in neutrophilia following dexamethasone (i.p.) administration. High-doses of systemically administered corticosteroids are not used in human subjects as this can result in serious side-effectssuppression of the hypothalamic-pituitary-adrenal axis, reduction in bone metabolism, thinning of the skin, growth retardation and glaucoma [33]. A combined administration of the corticosteroid, budesonide and a NO donor in the form of NCX 1020 may be of use in treating corticosteroid resistant COPD where high-doses may be toxic and not allowed. Acknowledgements We wish to thank Nicox S.A., Sophia Antipolis, France for financial support with a studentship to BJN. References [1] Murray CJ, Lopez AD. Global mortality, disability and the contribution of risk factors: global burden of disease study. Lancet 1997;349:1436–42. [2] Stockley RA. Neutrophils and the pathogenesis of COPD. Chest 2002; 121:151S– 5S. [3] Thompson AB, Daughton D, Robbins RA, Ghafouri MA, Oeklerking M, Rennard SI. Intraluminal airway inflammation in chronic bronchitis. Characterization and correlation with clinical parameters. Am Rev Respir Dis 1989;140:1527–37. [4] Keatings VM, Jatakanon A, Worsdell YM, Barnes PJ. Effects of inhaled and oral glucorticoids on inflammatory indices in asthma and COPD. Am J Respir Crit Care Med 1997;155:542–8. [5] Pauwels RA, Buist AS, Calverley MA, Jenkins CR, Hurd SS. Global strategy for the diagnosis, management and prevention of chronic obstructive pulmonary disease. NHLBI/WHO global initiative for chronic obstructive lung disease (GOLD) Workshop Summary. Am J Respir Crit Care Med 2001;163:1256–76. [6] Nevin BJ, Broadley KJ. Nitric oxide in respiratory disease. Pharmacol Therap 2002;95:259 –93. [7] Lindeman KS, Aryana A, Hirshman CA. Direct effects of inhaled nitric oxide on canine peripheral airways. J Appl Physiol 1995;78: 1898– 903. [8] Granger DN, Kubes P. Nitric oxide as anti-inflammatory agent. Meth Enzym 1996;269:434 –43. [9] Fox-Robichaud A, Payne D, Hasan SU, Ostrovsky L, Fairhead T, Reinhardt P, Kubes P. Inhaled NO as a viable antiadhesive therapy for ischaemia/reperfusion injury of distal microvascular beds. J Clin Invest 1998;101:2497–505. [10] Ansarin K, Chatkin JM, Ferreira IM, Gutierrez CA, Zamel N, Chapman KR. Exhaled nitric oxide in chronic obstructive pulmonary disease, relationship to pulmonary function. Eur Respir J 2001;17: 934–8. [11] Ichinose M, Suigura H, Yamagata S, Koarai A, Shirato K. Increase in reactive nitrogen species production in chronic obstructive pulmonary disease airways. Am J Respir Crit Care Med 2000;162:701– 6. [12] Baigorri F, Joseph D, Artigas A, Blanch L. Inhaled nitric oxide does not improve cardiac or pulmonary function in patients with an exacerbation of chronic obstructive pulmonary disease. Crit Care Med 1999;27:2153 –8. [13] Toward TJ, Broadley KJ. Airway reactivity, inflammatory cell influx and nitric oxide in guinea-pig airways after lipopolysaccharide inhalation. Br J Pharmacol 2002;131:271–81.

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