Giuseppina Bertorelli

Alberto Pesci

Silvia Peveri

Mario Mergoni

Attilio Corradi

Anna Maria Cantoni

Giovanni Tincani

Antonio Bobbio

Michele Rusca

Paolo Carbognani

Attilio Corradi #

Anna Maria Cantoni #

Giovanni Tincani°

Antonio Bobbio°

Michele Rusca°

Paolo Carbognani°

Alpha glucocorticoid receptor expression in different experimental rat models of acute lung injury

Keywords: ARDS, lung inflammation, acute lung injury, barotrauma, glucocorticoid acquired resistance, glucocorticoid receptor α

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

inhibit the inflammatory pathways through two specific intracellular glucocorticoid receptors (GRs), named GRα and GRβ. The aim of our study was to evaluate the histologic evidence of inflammatory injury and the GRα uptake of resident and inflammatory cells in different experimental models of acute lung injury.

Methods:

We studied 4 groups of rats: three different experimental rat models of lung injury and a control group. The acute lung injury was caused by barotrauma (due to an overventilation), oleic acid injection and mechanical ventilation. Results were compared to non ventilated rat control group. The duration of mechanical ventilation was of 2.5 hours. At the end of each experiment, rats were sacrificed. Lung biopsies were evaluated for morphologic changes. The immunohistochemistry was performed to study GRα expression.

Results: Histologic evidence of lung injury (alveolar and interstitial edema, vascular congestion, alveolar haemorrhage, emphysema, number of interstitial cells and neutrophils, and destruction of alveolar attachments) were present in all ventilated groups. Barotrauma lead to an additional inflammatory response. GRα expression significantly increased in the three ventilated groups compared with non ventilated groups. GRα expression was highest in barotrauma group.

A c c e p t e d m a n u s c r i p t INTRODUCTION

Acute respiratory distress syndrome (ARDS) is a term applied to a relatively specific morphologic lesion of multifactorial aetiology known as diffuse alveolar damage (DAD) [START_REF] Katzenstein | Diffuse alveolar damage -the role of oxygen, shock, and related factors. A review[END_REF]. It represents the extreme end of a spectrum of lung injury. The initiating events that lead to ARDS and its less severe manifestation, acute lung injury (ALI), are clinically widespread and diverse [START_REF] Wallace | Pathogenesis of acute microvascular lung injury and the acute respiratory distress syndrome[END_REF]. At presentation, early ARDS manifests with severe, diffuse, and nonhomogenous acute host inflammatory response of the pulmonary lobules, leading to a breakdown in the barrier and gas exchange function of the lung [START_REF] Steinberg | Acute lung injury and acute respiratory distress syndrome. The clinical syndrome[END_REF].

Despite apparent improvement in management and outcome of ARDS, the mortality rate of ARDS remains high, ranging from 35% to 65% [START_REF] Milberg | Improved survival of patients with acute respiratory distress syndrome (ARDS): 1983-1993[END_REF][START_REF] Suchyta | Increased survival of ARDS patients with severe hypoxemia (ECMO criteria)[END_REF]. Mechanical ventilation delays mortality in many patients with acute respiratory failure and is used to maintain adequate systemic oxygenation and to rest the respiratory muscles. However, over the last 2 decades, it has become evident that mechanical ventilation itself can augment or cause ALI. Recent experimental studies in various animal models have provided 3 lines of evidence suggesting that mechanical ventilation can initiate or exacerbate an inflammatory response: 1) pathologic evidence of neutrophil infiltration (6-7), 2) increased cytokine levels in lung lavage [START_REF] Tremblay | Injurious ventilatory strategies increase cytokines and c-fos m-RNA expression in an isolated rat lung model[END_REF] and 3) increased cytokine levels in the systemic circulation [START_REF] Chiumello | Mechanical ventilation affects local and systemic cytokines in an animal model of ARDS[END_REF][START_REF] Von Bethmann | Hyperventilation induces release of cytokines from perfused mouse lung[END_REF].

There are several experimental research models to construct a pathophysiological state of ARDS, attempting to fit pathogenesis (biology) with morphological (pathology) and clinical (physiology) findings (11). Intravenous injection of pure oleic acid into several different animal species has been observed to cause respiratory distress [START_REF] Dickey | Oleic-acid-induced lung injury in the rat. Failure of indomethacin treatment or complement depletion to ablate lung injury[END_REF]. Also barotrauma is characterized by the release of inflammatory mediators from cells within the lung. In animals, ventilation with the use of large tidal volumes caused the disruption of pulmonary epithelium and endothelium, lung inflammation, atelectasis, hypoxemia, and the release of inflammatory mediators [START_REF] Tremblay | Injurious ventilatory strategies increase cytokines and c-fos m-RNA expression in an isolated rat lung model[END_REF][START_REF] Tsuno | Histopathologic pulmonary changes from mechanical ventilation at high peak airway pressures[END_REF][START_REF] Parker | Mechanisms of ventilator-induced lung injury[END_REF][START_REF] Dreyfuss | Intermittent positive-pressure hyperventilation with high inflation pressures produces pulmonary microvascular injury in rats[END_REF][START_REF] Webb | Experimental pulmonary edema due to intermittent positive pressure ventilation with high inflation pressures: protection by positive end-expiratory pressure[END_REF][START_REF] Kolobow | Severe impairment in lung function induced by high peak airway pressure during mechanical ventilation: an experimental study[END_REF]. Biotrauma is the response to barotrauma characterized by the release of inflammatory mediators from cells within the lung [START_REF] Tremblay | Ventilator-induced injury: from barotrauma to biotrauma[END_REF]. This may help explain why most patients who die with ARDS succumb not because their lungs fail, but because of the development of multiple organ dysfunction syndrome (MODS) involving both the lungs and other organs [START_REF] Slutsky | Multiple system organ failure. Is mechanical ventilation a contributing factor?[END_REF][START_REF] Acute | Ventilation with lower tidal volumes as compared with traditional tidal volumes for acute lung injury and the acute respiratory distress syndrome[END_REF]. The aim of our study was to evaluate, in lungs of rats with different kinds of ALI (mechanical ventilation, oleic acid injection, barotrauma), the histologic changes and the GRα uptake of resident and inflammatory cells.

A c c e p t e d m a n u s c r i p t MATERIAL AND METHODS

We studied 45 male Wistar rats weighing from 300 to 600 g.

Anaesthesia and preparation

Subcutaneous injection of sodium thiopentale was used to induce (13mg/100gr) and maintain (10mg/100gr every 30 minutes) anaesthesia. The animals were placed in the supine position and were intubated through a cervical tracheostomy with a 14-gauge Teflon catheter which was secured by a ligature around the trachea. Before starting mechanical ventilation pancuronium bromide (0.1mg/kg) in iterated doses was administered intravenously for muscle relaxation. The animals were ventilated with a Harvard apparatus rodent volume ventilation (model 683, Harvard Apparatus, South Natick, MA) at a respiratory rate of 60 strokes/minute. Airway pressure was measured through a side port in the tracheal tube using a pressure transducer (Abbott Transpac II, Abbott Laboratories, Illinois) connected to a 4 channel monitor (Siemens Sirecust 1281, Siemens, Germany). The carotid artery and the jugular vein were isolated and catheterized at the neck respectively for arterial blood gases determination and direct intravenously normal saline solution (7 ml/Kg/h) and oleic acid in selected animals.

Animals were divided into 4 groups:

1. Non ventilated control group: 4 animals were immediately sacrificed and both lungs were excised through a median sternotomy.

2. Ventilated group: 13 animals were ventilated with a tidal volume of 10/ml/Kg and at a respiratory rate of 60 strokes/minute.

A c c e p t e d m a n u s c r i p t

3. Lung injury induced by oleic acid group: 19 rats were ventilated with a tidal volume of 10/ml/Kg and at a respiratory rate of 60 strokes/minute. Lung injury was induced by oleic acid injection. We have used the same model described by other authors [START_REF] Dickey | Oleic-acid-induced lung injury in the rat. Failure of indomethacin treatment or complement depletion to ablate lung injury[END_REF].

Lung injury was induced using oleic acid (0.2 ml/kg) that was dissolved in an equal volume of absolute alcohol and infused by a motor syringe during a 30-min period. To counteract circulatory instability caused by the oleic acid, 15 ml/kg of dextran (PM 70.000 daltons) in normal saline was infused over 30 min before the administration of oleic acid. The overall duration of mechanical ventilation in groups 2, 3 and 4 was of 2.5 hours. At the end of each experiment, all the animals were sacrificed by an intracardiac injection of potassium chloride and both of the lungs were removed through a median sternotomy.

Anatomic, histologic and immunohistochemical studies

The weight of each lung excised (expressed as weight of the lungs to weight of the rat ratio)

was measured. Lung biopsies were collected from the rats of all groups and evaluated for morphologic changes. Lungs for histologic examination were inflated to 30 cm H 2 O twice and then to 10 cm H 2 O. Lung biopsies were fixed in phosphate-buffered formalin and embedded in paraffin. Five µm thick sections were performed and stained for routine histology (H&E, Masson's Thricomic method, Giemsa) and immunohistochemistry.

Immunohistochemistry was performed as previously described (24-26) in all biopsies with an indirect avidin-biotin complex immunoperoxidase method (Vectastasin Elite PK 6100, Vector, CA, USA) using a mouse-rabbit secondary antibody (LSAB code n° K0675, DakoCytomation, Denmark) and 3,3 diamminobenzidine (DAB code n° K4368, DakoCytomation, Denmark) as chromatic substrate. The following primary antibody was used: polyclonal rabbit anti-glucocorticoid receptor α antibody (Bioaffinity Reagents, Golden, Colo), 1/100, also using a microwave antigen retrieval step in citrate buffer 10 mM at pH 6.0.

A c c e p t e d m a n u s c r i p t

For negative control preparations, the primary antibody was replaced by either non-specific rabbit immunoglobulin or Tris-buffered saline solution.

Assessment and quantification of histologic staining

Light microscopy was performed using a Olympus microscope (Olympus BH-2) at a magnification of X100. Twenty random fields were examined in each lung biopsy. Biopsies were scored for injury severity from 0 to 3 (with 0 meaning normal and 3 indicating the most severe injury) for the following histologic changes: alveolar and interstitial edema, vascular congestion, alveolar haemorrhage and emphysema. Specimens were scored for injury severity by a researcher blinded to the different groups.

Twenty random fields were examined at X1000 for the destruction of alveolar attachments.

Alveolar attachments were identified as the alveolar septa that extend from the outer wall of the nonrespiratory bronchioles. Those attachments showing rupture or discontinuity were defined as destroyed alveolar attachments. The number of destroyed alveolar attachments was expressed as a percentage over the total number of alveolar attachments.

In addition, 20 random fields were examined at X1000 for the number of interstitial cells and the number of neutrophils (identified by their characteristic nucleus multilobulated and hyperchromic without nucleoli) were counted. Cells were expressed as cells/HPF.

Assessment and quantification of immunohistochemical staining

For each biopsy, all anti-glucocorticoid receptor α positively stained cells were counted in 20 random HPF at the magnification of X1000. Cells were expressed as GRα positive cells/HPF.

Statistical analysis

Non-parametric statistical tests (Mann-Whitney U test) were used in the analysis. The median values and range are presented in the text and the accompanying figures and tables. Correlation coefficients were obtained by Spearman's rank-order method. In all cases a value of p< 0.05 was considered significant.

A c c e p t e d m a n u s c r i p t

RESULTS

The weights of the rats' lungs are reported in Fig. 1 Histologic evaluation results

Histologic evidence of lung injury was present in all ventilated groups. Within 2.5 hours after starting ventilation there were notable histologic changes, such as alveolar and interstitial edema, vascular congestion, alveolar haemorrhage, emphysema (tab 1), destruction of alveolar attachments, increased number of interstitial cells and extensive infiltrate of neutrophils (tab 2) (Fig. 2b and2c). Histologic examination of the lungs demonstrated differences in lung injury among the groups. Lung injury was patchy in all groups. There were fewer areas of normal lung in the barotrauma group. There was more lung inflation in the lungs of rats ventilated with the highest tidal volume than in other groups. In groups 2, 3 and 4, there was a significant increase in the number of interstitial cells, neutrophils and destruction of alveolar attachments compared to group 1. The cell accumulation was highest in barotrauma group (Fig. 3a, Fig. 3b and Fig. 3c).

Immunohistochemical staining results

Immunohistochemical staining was performed successfully. GRα expression significantly increased in the three ventilated groups compared with non ventilated groups and was highest in the barotrauma group (Fig 4a and4b). The increased numbers of cells expressing GRα were mainly a reflection of an increase in infiltrating inflammatory cells and the number of GRα+ cells was strongly correlated with the number of neutrophils. However, also some resident cells showed an increased number of cells demonstrating GRα expression.

The number of GRα+ cells was strongly correlated with the number of neutrophils (p<0.0001, r=0.65) (Figure 6a). We observed also a significant correlation between the number of neutrophils with the weight of the lung of rats (p=0.006, r=0.43) (Figure 6b) and the number of GRα+ cells with the weight of the lung of rats (p<0.009, r=0.42) (Figure 6c).

A c c e p t e d m a n u s c r i p t

DISCUSSION

In our experimental models, the histologic evaluation showed the main characteristics of ARDS syndrome with diffuse inflammation and increased microvascular permeability. In particular, these findings were prominent in lung injury induced by barotrauma. We can conclude that our experimental models are effective in the ARDS inflammation assessment.

Our data showed that acute lung injury is associated with up-regulation of the inflammatory response and GRα overexpression. Barotrauma is the most effective mechanism inducing acute lung inflammation and GRα overexpression.

Histologic changes are in accordance with literature findings. ARDS represents a classic example of a disease state in which the potential for tissue repair and the potential for tissue injury balance is shifted dramatically towards excessive tissue injury. This inflammatory response gives rise to significant pulmonary endothelial and epithelial cell injury.

The acute lung damage following intravenous infusion of oleic acid has been studied as a model for the so-called chemical phase of post traumatic pulmonary fat embolism. Both the physiological and histological findings have been well studied. Despite the extensive work that has been done in examining oleic-acid-induced pulmonary changes, the precise mechanism of injury remain unclear [START_REF] Dickey | Oleic-acid-induced lung injury in the rat. Failure of indomethacin treatment or complement depletion to ablate lung injury[END_REF].

The adverse consequences of mechanical ventilation were traditionally thought to be due Failure to suppress inflammation could be due to tissue resistance to GCs or inadequacy of the level and duration of endogenous GC elevation to suppress host inflammatory response [START_REF] Annane | A 3-level prognostic classification in septic shock based on cortisol levels and cortisol response to corticotropin[END_REF]. GC-mediated resistance was originally described as a primary inherited familial syndrome, [START_REF] Chrousos | Syndromes of glucocorticoid resistance[END_REF][START_REF] Lamberts | Cortisol receptor resistance: the variability of its clinical presentation and response to treatment[END_REF] and was recently recognized as an acquired condition. Several in vitro studies have shown that cytokines may induce resistance to GCs by reducing GR binding affinity to cortisol [START_REF] Almawi | Abrogation of glucocorticoid-mediated inhibition of T cell proliferation by the synergistic action of IL-1, IL-6, and IFN-gamma[END_REF][START_REF] Kam | Combination IL-2 and IL-4 reduces glucocorticoid receptor-binding affinity and T cell response to glucocorticoids[END_REF][START_REF] Spahn | A novel action of IL-13: induction of diminished monocyte glucocorticoid receptor-binding affinity[END_REF]. Such abnormalities of GR function were demonstrated in T cells incubated with a combination of IL-2 and IL-4 (36), IL-1β, IL-6, interferon (INF)-γ (35) and IL-13 [START_REF] Kam | Combination IL-2 and IL-4 reduces glucocorticoid receptor-binding affinity and T cell response to glucocorticoids[END_REF]. GC resistance was induced in a cytokine concentration-dependent fashion and was reversed by the removal of cytokines [START_REF] Kam | Combination IL-2 and IL-4 reduces glucocorticoid receptor-binding affinity and T cell response to glucocorticoids[END_REF].

A c c e p t e d m a n u s c r i p t

GR-mediated resistance in the presence of systemic inflammation was also studied in experimental models of sepsis and sepsis-induced ARDS. In a sheep model of sepsisinduced ARDS (38-40), maximal binding capacity of GR decreased continuously after endotoxin infusion, while there was a marked elevation of cortisol levels [START_REF] Baeuerle | NF-kappa B as a frequent target for immuno-suppressive and anti-inflammatory molecules[END_REF]. The reduced GR binding correlates negatively with phospholipase A 2 activity, a gene that is stimulated by NF-kB. In a rat model of septic shock, GR blockade by minfepristone exacerbated the physiologic and pathologic changes induced by endotoxemia [START_REF] Bamberger | Molecular determinants of glucocorticoid receptor function and tissue sensitivity to glucocorticoids[END_REF]. Phospholipase A 2 activity in rats with 80% GR blockade was more marked than in those with 50% GR blockade [START_REF] Bamberger | Molecular determinants of glucocorticoid receptor function and tissue sensitivity to glucocorticoids[END_REF].

Monocytes taken from patients with sepsis developed near total GC resistance in vitro when cytokines, especially IL-2, were added (38). Several inflammatory cytokines, including TNFα, IL-1β, and IL-6 activate NF-kB [START_REF] Baeuerle | NF-kappa B as a frequent target for immuno-suppressive and anti-inflammatory molecules[END_REF]. It has been suggested that when cytokine-activated NF-kB forms protein-protein complexes with activated GR, the availability and activity of effective GR molecules are reduced [START_REF] Bamberger | Molecular determinants of glucocorticoid receptor function and tissue sensitivity to glucocorticoids[END_REF][START_REF] Barnes | Glucocorticoid resistance in asthma[END_REF]. This functional reduction in GR availability is associated with an increased number of GR [START_REF] Bamberger | Molecular determinants of glucocorticoid receptor function and tissue sensitivity to glucocorticoids[END_REF][START_REF] Barnes | Glucocorticoid resistance in asthma[END_REF].

The increase in GRβ isoform expression is considered responsible for glucocorticoid resistance. However, in this case we did not observe any increase in the production of GRβ. related to resistance to GCs observed in different models of ALI. The lack of change in GRß expression is interesting as neutrophils have previously been reported to express high levels of GRß in man [START_REF] Strickland | High constitutive glucocorticoid receptor β in human neutrophils enables them to reduce their spontaneous rate of cell death in response to corticoids[END_REF][START_REF] Xu | Serine-arginine-rich protein p30 directs alternative splicing of glucocorticoid receptor pre-mRNA to glucocorticoid receptor β in neutrophils[END_REF]. This could reflect the possibility that in rat GRß has a distinct function compared to that in human. In this regard, we know that some animal models of systemic glucocorticoid resistance such as New World monkeys have approximately 10-fold higher GRß than GRα level [START_REF] Vottero | Glucocrticoid receptor beta: view I[END_REF] and, on the contrary, mice, known to be extremely steroidsensitive animals, do not appear to have GRß [START_REF] Otto | Absence of glucocorticoid receptor β in mice[END_REF].

We observed an increased number of GRα positive cells related to the increased number of neutrophils. However, neutrophils have previously been expressed in high levels of GRß in man [START_REF] Strickland | High constitutive glucocorticoid receptor β in human neutrophils enables them to reduce their spontaneous rate of cell death in response to corticoids[END_REF][START_REF] Xu | Serine-arginine-rich protein p30 directs alternative splicing of glucocorticoid receptor pre-mRNA to glucocorticoid receptor β in neutrophils[END_REF]. This could reflect the possibility that in rat GRß has a distinct function compared to that in humans. In this regard, we know that some animal models of systemic glucocorticoid resistance such as New World monkeys have approximately 10-fold higher GRß than GRα levels [START_REF] Vottero | Glucocrticoid receptor beta: view I[END_REF] and, on the contrary, mice, known to be extremely steroidsensitive animals, do not appear to have GRß [START_REF] Otto | Absence of glucocorticoid receptor β in mice[END_REF].

A c c e p t e d m a n u s c r i p t

The concept of acquired resistance can explain the significantly increased number of GRα positive cells that we found in the three ventilated groups compared with the non ventilated groups. In the barotrauma group, where the inflammatory changes were prominent, the number of GRα was highest.

A c c e p t e d m a n u s c r i p t

Figure captions

Fig. 1 The weights of the rats' lungs in ventilated, oleic acid and barotrauma groups. Rupture of septal walls is also present (arrow points). Original magnification x200.

4 .

 4 Lung injury induced by barotrauma group: in 9 animals the injury was induced by ventilation volumes that reached a plateau pressure of 40 cm H 2 O at a respiratory rate of 60 strokes/minutes.

A c c e p t e d m a n u s c r i p t

  largely to mechanical factors, such as haemodynamic compromise from decreased venous return and/or barotrauma due to overdistension of the lung. More recently research has revealed more subtle types of ventilator-induced lung injury including diffuse alveolar damage and up-regulation of the inflammatory response. The latter is referred to as biotrauma. Ranieri et al. showed that mechanical ventilation can induce a cytokine response that may be attenuated by a strategy to minimize overdistension and recruitment/derecruitment (27). Experimental animal data clearly demonstrate that overstretching of lung cells and/or allowing recruitment/derecruitment of the lung can lead to an increase in lung cytokines[START_REF] Tremblay | Injurious ventilatory strategies increase cytokines and c-fos m-RNA expression in an isolated rat lung model[END_REF][START_REF] Von Bethmann | Hyperventilation induces release of cytokines from perfused mouse lung[END_REF]28). Ventilation-induced chemokine and cytokine release is associated with activation of NF-kB. Under conditions in which there is increased lung permeability, these cytokines may translocate from the alveolar space to the systemic circulation.When we compared histologic changes of our two different experimental models (oleic acid and barotrauma), we found that biotrauma is the most effective model to obtain pathologic evidence of inflammation and increased microvascular permeability with an influx of large numbers of neutrophils into the lung. It is now believed that mediators derived from inflammatory cells create an injurious response directed against the microvasculature of the lung. The resultant injury leads to disruption of the alveolar-capillary interface and leakage of a protein-rich inflammatory exudate into the airspace. The inflammatory cell primarily implicated in this process is the neutrophil.Glucocorticoids are the most important natural inhibitors of inflammation (29,30). However, endogenous GCs are not always effective in suppressing ARDS inflammation. The failure to improve in ARDS (unresolving ARDS) is frequently associated with the activated GRs to downregulate the transcription of inflammatory cytokines despite elevated levels of circulating cortisol, a condition defined as systemic inflammation-associated acquired GC resistance and potentially reversible with prolonged GC supplementation. Meduri GU et al.have shown that, in patients with unresolving ARDS, normal peripheral blood leukocytes exposed to plasma samples collected during prolonged methylprednisolone treatment exhibited a progressive increase in cytoplasmic binding of GRα to NF-kB and a concomitant reduction in NF-kB binding to DNA and transcription of TNFα and the IL-1β[START_REF] Meduri | Prolonged methylprednisolone treatment suppresses systemic inflammation in patients with unresolving acute respiratory distress syndrome: evidence for inadequate endogenous glucocorticoid secretion and inflammationinduced immune cell resistance to glucocorticoids[END_REF].

A c c e p t e d m a n u s c r i p t A c c e p t e d m a n u s c r i p t 21 .

 21 Franchimont D, Kino T, Galon J, Meduri GU, Chrousos G. Glucocorticoids and inflammation revisited: the state of the art. NIH Clinical Staff Conference. Neuroimmunomodulation. 2003;10:247-260. 22. Baeuerle PA, Baltimore D. Activation of DNA-binding activity in an apparently cytoplasmic precursor of the NF-kappa B transcription factor. Cell. 1988; 53:211-217. 23. Oakley RH, Sar M, Cidlowski JA. The human glucocorticoid receptor beta isoform. Expression, biochemical properties, and putative function. J Biol Chem. 1996;271:9550-9559. 24. Bocchino V, Bertorelli G, Bertrand CP, Ponath PD, Newman W, Franco C, Marruchella A, Merlini S, Del Donno M, Zhuo X, Olivieri D. Eotaxin and CCR3 are up-regulated in exacerbations of chronic bronchitis. Allergy. 2002; 57:17-22. 25. Bertorelli G, Bocchino V, Zhou X, Zanini A, Bernini MV, Damia R, Di Comite V, Grima P, Olivieri D. Dendritic cell number is related to IL-4 expression in the airways of atopic asthmatic subjects. Allergy. 2000; 55:449-454. 26. Bocchino V, Bertorelli G, Zhuo X, Grima P, Di Comite V, Damia R, Chetta A, Del Donno M, Foresi A, Casalini A, Testi R, Olivieri D. Short-term treatment with a low dose of inhaled fluticasone propionate decreases the number of CD1a+ dendritic cells in asthmatic airways. Pulm Pharmacol Ther. 1997;10:253-259. 27. Ranieri VM, Suter PM, Tortorella C, De Tullio R, Dayer JM, Brienza A, Bruno F, Slutsky AS. Effect of mechanical ventilation on inflammatory mediators in patients with acute respiratory distress syndrome. A randomized controlled trial. JAMA 1999; 282:54-61. 28. Held HD, Boettcher S, Hamann L, Uhlig S. Ventilation-induced chemokine and cytokine release is associated with activation of NF-kB Am J Respir Crit Care Med 2001; 163:711-716. 29. Chrousos GP. The hypothalamic-pituitary-adrenal axis and immune-mediated inflammation. N Engl J Med. 1995; 332:1351-1362.

Fig. 2 a

 2 Fig. 2 a) Hematoxylin-eosin staining. Microphotograph from control animals. b) Hematoxylin-eosin staining. Microphotograph showing an intense rupture of septal walls in a rat ventilated with high inflation flow (arrows). Original magnification x100. c) Hematoxylin-eosin staining. Microphotograph showing an intense cellular infiltration within the alveolar walls from a rat ventilated with high inflation flow. There is interstitial and intraalveolar edema (solid line arrows) and some haemorrhage (intermitted line arrows).

Fig. 3 Number

 3 Fig.3 Number of interstitial cells (a), neutrophils (b) and destruction of alveolar attachments (c) in the lungs of ventilated, oleic acid and barotrauma groups compared to control group.

Fig. 4 Immunohistochemistry

 4 Fig.4 Immunohistochemistry staining of lung tissue for expression of GRα. Representative images of GRα immunostaining in lung preparations from non-ventilated rat lung (a) compared with rat lung from barotrauma group (b). Note the increased number of GRα immunoreactive cells in alveolar walls of the lung from barotrauma rat group (b) (brown spots) compared with the lung from non-ventilated rat group (a) (brown spots indicated by arrows).

Fig. 5 GRα

 5 Fig. 5 GRα (a) expression in the lungs of three ventilated experimental groups compared with non ventilated group.

Fig. 6 Correlation

 6 Fig. 6Correlation between the number of GRα+ cells and the number of neutrophils (a), the number of neutrophils with the weight of the lung of rats (b) and the number of GRα+ cells with the weight of the lung of rats (c).

A c c e p t e d m a n u s c r i p t Figure 1 A c c e p t e d m a n u s c r i p t Figure 2 A c c e p t e d m a n u s c r i p t A c c e p t e d m a n u s c r i p t

 12 Figure 1

Figure 3 control

 3 Figure 3

Figure 4 A c c e p t e d m a n u s c r i p t

 4 Figure 4

  Figure 5

  Figure 6

  The animals were treated in compliance with the Principles of Laboratory Animal Care formulated by the Institute of

	Laboratory Animal Resources, the Guide for the Care and Use of Laboratory Animals
	published by the National Institute of Health (NIH Publication No. 86-23, revised in 1985)
	and the Guide to the Care and Use of Experimental Animals formulated by the Canadian
	Council of Animal Care (CCAC 2 nd Ed 1993).

Table 2 -Histologic evaluation results Parameters (HPF)

 2

		Non Ventilated	Ventilated	Oleic Acid	Barotrauma
		Controls	Group	Group	Group
	Interstitial
	cells	17.90	39.80	32.50	27.40
		(17.50-21.00)	(24.00-49.60)	(22.40-44.60)	(22.50-65.50)
	Neutrophils	0.85	1.20	1.80	3.60
		(0.80-0.90)	(1.00-1.90)	(0.50-3.80)	(2.80-7.50)
	Destruction of
	alveolar	11.75	18.70	20.60	17.20
	attachments	(10.80-14.20)	(13.40-26.30)	(13.10-26.40)	(14.80-24.90)

Values were expressed as median (range)

Parameters (HPF)

Non Ventilated Controls Ventilated Group

Oleic Acid Group Barotrauma Group Vascular Congestion

Intraalveolar Edema

Intraalveolar Haemorrhage

Values were expressed as median (range)


J Moita

C Bárbara

J Cardoso

R Costa

M Sousa

J Ruiz

M L Santos

Tiotropium improves FEV 1 in patients with COPD irrespective of smoking status

Keywords: Tiotropium, Lung function, FEV 1, Smoking status, COPD

This study evaluated whether the effect of tiotropium on the change in trough forced expiratory volume in 1 second (FEV 1 ), vs. placebo, is affected by smoking status. In a 3month, double-blind study in 31 centres in Portugal, 311 (289 completed) patients were randomised to tiotropium 18 µg once daily or placebo. Baseline mean (standard deviation (SD)) FEV 1 was 1.11 (0.39) l in the tiotropium group and 1.13 (0.39) l in the placebo group.

Patients had an average smoking history of 55 (25.7) pack-years; 80 (26%) were smokers and 224 (74%) were ex-smokers. The primary end point was change in morning pre-dose (i.e. trough) FEV 1 after 12 weeks. Trough FEV 1 at 12 weeks was significantly improved with tiotropium vs. placebo: the difference in means was 102 ml, P=0.0011, 95% confidence interval (CI) (41, 164). The difference in means in smokers was 138 ml, P=0.0105, CI (32, 244); in ex-smokers it was 66 ml, P=0.0375, CI (3, 129). The difference between smokers and ex-smokers was not statistically significant (P=0.6982) and may be due to greater variability and differences in disease severity. The significant improvement in lung function in patients treated with tiotropium vs. placebo in both smokers and exsmokers suggests that tiotropium is an effective and well-tolerated therapy in chronic obstructive pulmonary disease (COPD), regardless of smoking status.

A c c e p t e d m a n u s c r i p t 1. Introduction

Cigarette smoking is a major risk factor for the development of chronic obstructive pulmonary disease (COPD) in susceptible individuals worldwide and may account for up to 90% of the risk in developed countries [1][START_REF] Bartal | COPD and tobacco smoke[END_REF][START_REF] Fletcher | The natural history of chronic airflow obstruction[END_REF]. Evidence suggests that the number of years spent as a smoker and amount of cigarettes smoked impacts on the prevalence of respiratory symptoms, exacerbations, rate of decline in lung function (i.e. forced expiratory volume in 1 second [FEV 1 ]) and COPD-related mortality [START_REF] Buist | Risk factors for COPD[END_REF][START_REF] Tashkin | The UCLA population studies of chronic obstructive respiratory disease: XI. Impact of air pollution and smoking on annual change in forced expiratory volume in one second[END_REF][START_REF] Becklake | The 'healthy smoker': A phenomenon of health selection?[END_REF][START_REF] Zalacain | Predisposing factors to bacterial colonization in chronic obstruction pulmonary disease exacerbation[END_REF]. Cigarette smoking is thought to exert its effects via increased inflammation, airway wall fibrosis, destruction of alveolar attachments, and inhibition of repair mechanisms [START_REF] Bartal | COPD and tobacco smoke[END_REF][START_REF] Willemse | Effect of 1-year smoking cessation on airway inflammation in COPD and asymptomatic smokers[END_REF].

Currently, smoking cessation is the only intervention shown to slow the long-term progression of airflow obstruction as measured by the decline in FEV 1 [1]. However, smoking cessation is far from easy and smoking cessation programmes generally have poor success rates [START_REF] Jorenby | A controlled trial of sustained-release bupropion, a nicotine patch, or both for smoking cessation[END_REF][START_REF] Tashkin | Smoking cessation in patients with chronic obstructive pulmonary disease: a double-blind, placebo-controlled, randomised trial[END_REF][START_REF] Silagy | Meta-analysis on efficacy of nicotine replacement therapies in smoking cessation[END_REF]. Evidence also suggests that chronic inflammation persists after smoking has stopped [START_REF] Hogg | Why does airway inflammation persist after the smoking stops?[END_REF], which will continue to contribute to lung function decline.

Hence, it is important to identify treatments that benefit patients with COPD, despite the fact that they might continue to smoke.

Tiotropium is a once-daily maintenance treatment for COPD that provides 24-hour efficacy due to prolonged M 3 -receptor antagonism. It has consistently been shown to improve lung function, exercise tolerance and health status, and reduce dyspnea in patients with COPD [START_REF] Casaburi | A longterm evaluation of once-daily inhaled tiotropium in chronic obstructive pulmonary disease[END_REF][START_REF] Vincken | 11 A c c e p t e d m a n u s c r i p t on behalf of the Dutch/Belgian Tiotropium Study Group. Improved health outcomes in patients with COPD during 1 yrs treatment with tiotropium[END_REF][START_REF] O'donnell | Effects of tiotropium on lung hyperinflation, dyspnoea and exercise tolerance in COPD[END_REF]. Post hoc analysis of two combined 1-year placebo trials has also provided preliminary evidence to suggest that long-term maintenance treatment with tiotropium once daily may slow the decline in trough (i.e. morning pre-dose) FEV 1 compared with placebo [START_REF] Anzueto | One-year analysis of longitudinal changes in spirometry in patients with COPD receiving tiotropium[END_REF]; a possibility that is currently specifically being investigated in a 4-year prospective trial [START_REF] Decramer | Clinical trial design considerations in assessing long-term functional impacts of tiotropium in COPD: the UPLIFT trial[END_REF].

The rationale for performing this trial was based on a meta-analysis from seven clinical trials with the shorter-acting anticholinergic, ipratropium, in patients with moderate to severe COPD, which suggested that the improvement in baseline lung function in ipratropium-treated patients was greater in ex-smokers than in smokers [START_REF] Rennard | Extended therapy with ipratropium is associated with improved lung function in patients with COPD. A retrospective analysis of data from seven clinical trials[END_REF]. The aim of this study, entitled 'Spiriva ® Assessment of FEV 1 ' (SAFE), was to evaluate whether the effect of tiotropium on the change in trough FEV 1 is affected by smoking status.

A c c e p t e d m a n u s c r i p t 2. Methods

Study design

This was a randomised, double-blind, parallel-group, placebo-controlled, 3 month study conducted in 31 centres in Portugal. The study (#205.282) was designed to determine whether the effect of tiotropium on trough FEV 1 in patients with COPD was affected by smoking status. The study was approved by regulatory and ethics committees at all centres.

Subjects

Males or females aged ≥40 years with a diagnosis of COPD (FEV 1 ≤70% of predicted and FEV 1 /forced vital capacity (FVC) ≤70% [START_REF] Celli | Standards for the diagnosis and care of patients with chronic obstructive pulmonary disease[END_REF]) and a smoking history of ≥10 pack-years were eligible for inclusion.

Patients were asked about their smoking status on the first visit. Smoking status consisted of two categories: smokers and ex-smokers (0 cigarettes/day).

Patients were not included if they had a history of asthma, allergic rhinitis, atopy, myocardial infarction, unstable arrhythmia, or if they had any clinically significant disease that might put the patient at risk because of study participation. Patients with ≥3 exacerbations of COPD in the preceding year or an exacerbation or lower respiratory tract infection within the 6 weeks prior to randomisation were also excluded.

Concomitant use of prn salbutamol MDI (100 µg/puff; withheld for at least 6 hours prior to each clinic visit), long-acting β 2 -agonists and continued use of theophylline preparations (excluding 24-hour preparations) (both withheld for at least 24 hours prior to each clinic visit) were allowed during the study period. Concomitant use of mucolytics, orally inhaled corticosteroids, minimal doses of oral corticosteroids (equivalent to prednisone ≤10 mg/day or ≤20 mg/alternate days) were allowed if the dosage was stabilized for at least 6 weeks before the study. Temporary increases in the dose of theophylline preparation of ≤7 days or addition/increased dose of oral steroids for ≤2 weeks were allowed for the treatment of an exacerbation during the study period. If appropriate, scheduled visits were postponed for at least 1 week, but not more than 2 weeks. Use of antibiotics was not restricted. Short-acting anticholinergics, oral β 2 -agonists, antileukotrienes, and other investigational drugs were not allowed during the study.

A c c e p t e d m a n u s c r i p t 2.3. Assessments

After a 2-week run-in period, patients were randomised to receive tiotropium 18 µg once daily, delivered via the HandiHaler ® device (Boehringer Ingelheim, Ingelheim am Rhein, Germany), or placebo for 12 weeks. FEV 1 and FVC were measured on test days at 10 minutes (±5 minutes) prior to administration of study drug (trough). Measurements were performed in triplicate using a Datospir 120C spirometer (Sibelmed, Barcelona, Spain) in accordance with American Thoracic Society criteria [START_REF]Standardization of Spirometry, 1994 Update[END_REF].

The primary end point was the change in trough FEV 1 after 12 weeks of treatment.

Trough FEV 1 was measured 24 hours after the previous dose of study drug on Day 1 (randomisation) as well as after 6 and 12 weeks of treatment. Secondary end-points included trough FEV 1 after 6 weeks of treatment, trough FVC after 6 and 12 weeks of treatment, assessment of COPD symptoms, Physician's Global Evaluation, Quality of Life Questionnaire (EQ-5D) and use of daytime and night-time rescue medication (salbutamol MDI 100 µg/puff). Rescue medication use, cigarette consumption and drug compliance were recorded in patient diary cards. Adverse events were collected throughout the study.

Statistical analysis

For the primary end point, the comparison between tiotropium and placebo was assessed via an analysis of covariance (ANCOVA) model, with treatment and centre as fixed effects, and mean trough FEV1 baseline as covariate. The primary end point was also stratified according to smoking status (smokers and ex-smokers), which was added to the ANCOVA model as a fixed effect. The least square mean (LSM) for FEV 1 response was computed and compared for each treatment group, stratified by smoking status, The secondary spirometry measures were also analyzed using a similar ANCOVA model. The COPD symptoms and global evaluation by investigator were analysed only descriptively.

The EQ-5D questionnaire was evaluated following EuroQol Group Enterprise conventions [START_REF] Badia | The Spanish version of EuroQol: a description and its applications. European Quality of Life scale[END_REF]. Descriptive statistics were used for safety variables.

Efficacy analyses were performed using the Full Analysis Set (FAS), which included all patients who received at least one dose of treatment and had at least a baseline value and who did not change smoking status during the trial. The Safety Analysis Set (SAS) included all patients who received at least one dose of treatment and had one safety evaluation after treatment.

A c c e p t e d m a n u s c r i p t

To detect a difference of 130 ml with 90% power based on a SD of 215 ml, 59

patients were required in each treatment group. In order to obtain 59 ex-smokers in the placebo group, based on assumed ratio of smokers to ex-smokers of 3:2, a total of 148 patients were required in each treatment group.

Results

Subjects

A total of 335 patients were screened and 311 were randomised to treatment.

Seven patients changed their smoking status during the 12 weeks after randomisation, and were excluded from the study. A total of 304 patients were included in the Full Analysis Set (tiotropium: 144; placebo: 160) (Table 1). The baseline characteristics for the remaining 304 patients were comparable across treatment groups (Table 1).

Patients had a mean smoking history of 55 pack-years, and 80 (26%) were smokers and 224 (74%) were ex-smokers. A greater proportion of ex-smokers (27%) had very severe disease (defined as FEV 1 /FVC <70% and FEV 1 <30% predicted) compared with smokers (19%). In the tiotropium treatment group, the study was unable to provide smoker and ex-smoker groups with comparable baseline characteristics for smoking history and FEV 1 . Compared with ex-smokers in the tiotropium group, smokers in the tiotropium group had a higher pack-year smoking history (5.6 pack-years) and a higher FEV 1 (0.20 L). The percentage completing the study according to protocol was high at 92.9% (289/311) (Table 2).

Efficacy assessments

In the overall group population, tiotropium significantly improved the mean trough FEV 1 at 12 weeks compared with placebo, with a mean difference of 102 ml (P=0.001) (Table 3).

Compared with placebo, tiotropium significantly improved mean trough FEV 1 after 12 weeks in both smokers and ex-smokers though the variability was greater with smokers (Figure 1). At study end, the mean difference between the tiotropium and placebo groups was 138 ml (P=0.011) in the smokers group, and 66 ml (P = 0.038) in the ex-smokers group. The difference between smokers and ex-smokers was not statistically significant (P=0.698).

A c c e p t e d m a n u s c r i p t

A similar trend was also observed for trough FEV 1 at 6 weeks. The mean difference between the tiotropium and placebo groups was 94 ml (95% CI: 154, 34; P=0.022). The difference between treatment groups was 121 ml (95% CI: 226, 15; P=0.025) for smokers and 67 ml (95% CI: 124, 10; P=0.021) for ex-smokers. There was no evidence of tachyphylaxis (Figure 1).

The mean change from baseline in trough FEV 1 was generally higher in smokers than in ex-smokers.

Tiotropium significantly improved mean trough FVC after 12 weeks compared with placebo (P=0.019) (Table 3). Improvements compared with placebo occurred in both smokers and ex-smokers, though they were significant only in ex-smokers. However, the difference between responses to tiotropium in smokers and ex-smokers was not statistically significant (P=0.535).

Compared with placebo, patients in the tiotropium group used fewer doses of daytime and night-time rescue medication during the study. This difference tended to increase over the 12-week treatment period. The difference in daytime usage between placebo and tiotropium was significant at Weeks 2, 6, 8, 11 and 12 (P<0.05). The magnitude of the reduction in daytime rescue medication use with tiotropium compared with placebo tended to be greater in smokers than ex-smokers in the latter 6 weeks of treatment (Figure 2). Similar trends were seen with night-time rescue medication.

COPD symptoms, global evaluation by the investigator and the EQ-5D showed no differences between treatment groups.

Safety assessment

The proportion of patients experiencing at least one adverse event during the study was 11.6% in the tiotropium group and 15.9% in the placebo group. Six patients (4.1%) in the tiotropium group and three patients (1.8%) in the placebo group experienced a serious adverse event, including two deaths (both in the tiotropium group), but none of these nine events was considered to be related to study medication.

Respiratory system disorders were distributed in three different groups: lower (including COPD exacerbations), upper and other. The results showed that the most commonly reported adverse events in both the tiotropium and placebo groups were: Lower Respiratory disorders (18.3% and 20.1%, respectively); and Upper Respiratory disorders

A c c e p t e d m a n u s c r i p t

(5.4% and 4.9%, respectively) (Table 4).

Discussion

The aim of this study was to determine whether the effect of inhaled tiotropium on lung function in patients with COPD was affected by smoking status. The primary outcome of this study showed that there was a significant improvement in trough FEV 1 after 12 weeks' treatment with tiotropium compared with placebo, irrespective of smoking status.

These data were supported, at least in part, by the secondary outcomes.

The use of short-acting bronchodilators for symptomatic relief provides an indication of the degree of dyspnea experienced by the patient. Tiotropium reduced daytime and night-time rescue medication compared with placebo during the study, and the difference between active treatment and placebo tended to increase over the 12-week treatment period. Subgroup analysis suggests that the mean response to tiotropium for reduction of daytime use of rescue medication was significantly greater in smokers compared with ex-smokers. However, though a trend was evident at other time points, this result may be related to greater variability within these subgroups and differences in disease severity.

There have been few studies specifically analysing the relationship between bronchodilators and smoking status. A meta-analysis of seven trials in moderate to severe COPD has suggested that the bronchodilator response to the anticholinergic ipratropium may be greater in ex-smokers compared with smokers [START_REF] Rennard | Extended therapy with ipratropium is associated with improved lung function in patients with COPD. A retrospective analysis of data from seven clinical trials[END_REF]. Examination of the subgroup of ex-smokers in these trials showed them to have a baseline FEV 1 of about 20% less than smokers. Hence, the authors of the meta-analysis suggested that the greater response with ipratropium in ex-smokers might be related to more severe impairment of lung function. This result could have relevance in the interpretation of the Lung Health Study, which suggested no sustained additional effect of ipratropium on the reduced rate of decline in lung function as a result of a smoking cessation programme [START_REF] Anthonisen | Effects of smoking intervention and the use of an inhaled anticholinergic bronchodilator on the rate of decline of FEV 1 . The Lung Health Study[END_REF]. The Lung Health Study recruited only current smokers, which, in addition to the reported use of an average of two doses of ipratropium per day rather than the prescribed three to four doses per day, could have limited the effect of ipratropium compared with that which may occur in long-term ex-smokers.

Ideally, interventions in COPD need to be effective in both smokers and exsmokers, particularly as many patients find it difficult to stop smoking. However, smoking cessation must be viewed as the most important therapeutic intervention for those who showing beneficial effects of tiotropium treatment on lung function in both smokers and exsmokers are encouraging. A trend for greater responses with tiotropium in smokers, which is in contrast to the findings with ipratropium, may be suggested from some data in this trial. Data from smokers showed greater variability than data from ex-smokers and a greater proportion of ex-smokers had more severe disease than smokers, both of which may account for the lack of statistical significance between groups. More severe disease in ex-smokers was also shown in patients recruited in 1-year study trials with tiotropium [START_REF] Anzueto | One-year analysis of longitudinal changes in spirometry in patients with COPD receiving tiotropium[END_REF].

In this case, the authors speculated that patients with more rapidly progressing disease are more likely to have the incentive to stop smoking successfully compared with those with more gradual disease progression.

There are several limitations to the current study. First, according to the protocol, the proportions of smokers and ex-smokers were expected to be 60% and 40%, respectively. However, the proportions were modified to 32% smokers and 68% exsmokers in order to achieve at least 85% statistical power in the analyses of smokers. Due to enrolment difficulties the ratio of smokers to ex-smokers became more disproportionate.

As seven patients changed their smoking status during the study and were excluded from the analyses, a total of 80 smokers were included in the trial (26.3% instead of the expected 32%; 40 smokers per treatment arm), with this ratio the statistical power in the analyses of smokers was 75%. Consequently, the proportion of smokers available for analysis was lower than planned in the protocol, which may have increased the variability in the data in the smokers subgroup. Second, as smoking abstinence was not verified by saliva cotinine or expired carbon monoxide some patients may have been misclassified.

Third, the duration of 12 weeks may not be representative of the long-term effects of maintenance treatment with tiotropium in patients with COPD stratified by smoking status.

Hence, further data from large, long-term trials with tiotropium, such as the ongoing, 4-year Understanding Potential Long-term Impacts on Function with Tiotropium (UPLIFT) trial [START_REF] Decramer | Clinical trial design considerations in assessing long-term functional impacts of tiotropium in COPD: the UPLIFT trial[END_REF] are required to confirm these results.

In summary, tiotropium significantly improved lung function in both smokers and ex-smokers compared with placebo. This, combined with a favourable safety profile from this trial, suggests that tiotropium is an effective and well-tolerated therapy in COPD, regardless of smoking status. *Analysis includes all 311 patients (i.e, including the seven patients who changed their smoking status during the trial)

A c c e p t e d m a n u s c r i p t

A c c e p t e d m a n u s c r i p t

  continue to smoke and should be encouraged with the latest advances in treating nicotine addiction whenever feasible. Bronchodilators are considered first-line maintenance therapy in the management of symptomatic COPD [1]. Hence, the results of the current study

A c c e p t e d m a n u s c r i p t A c c e p t e d m a n u s c r i p t

Fig. 1 .

 1 Fig. 1. Mean change in trough FEV 1 during the 12-week study by treatment group and smoking status: (A) smokers and (B) ex-smokers.

Fig. 2 .

 2 Fig. 2. Mean change (tiotropium-placebo) in daytime rescue medication use in smokers and ex-smokers.

A c c e p t e d m a n u s c r i p t 409 410

 410

Table 1

 1 Demographic and baseline characteristics (full analysis set)

		Tiotropium (n=144)	Placebo (n=160)
		Non-smokers	Smokers	Non-smokers	Smokers
	Patients (n)	104	40	120	40
	Males (%)	97	93	95	93
	Age (years)*	65.7±8.6	61.6±9.8	65.7±9.0	64.0±7.2
	Duration of COPD	14.0±10.9	9.4±6.8	13.7±10.5	12.3±8.5
	(years)*
	Smoking history (pack-	54.3±27.1	59.9±23.4	54.3±26.7	55.3±17.3
	years)*
	Duration of smoking	9.1±9.8	0.0±0.0	10.2±10.4	0.0±0.0
	cessation (years)*
	Median (range)	5.6 (0.0-51.0)	0.0±0.0	6.1 (0.0-55.0)	0.0±0.0
	duration of smoking
	cessation (years)
	FEV 1 (L)*	1.06±0.37	1.26±0.42	1.13±0.40	1.15±0.40
	FEV 1 (% predicted)*	38.4±12.8	44.4±13.9	42.3±15.3	40.4±14.5
	FVC (L)*	2.42±0.76	2.68±0.73	2.52±0.69	2.54±0.71
	FEV 1 /FVC (%)*	44.4±11.0	47.9±13.7	45.2±11.5	46.7±13.4
	*Mean (SD); FEV 1 , forced expiratory volume in 1 second; FVC, forced vital capacity

Table 2

 2

	Disposition of patients
		Tiotropium	Placebo	Total
	_____________________________________________________________________
	Patients randomised (%)	147(100.0)	164(100.0)	311(100.0)
	Completed study according
	to protocol	136(92.5)	153(93.3)	289(92.9)
	Reasons for premature
	discontinuation:
	Unexpected worsening of
	COPD	1(0.7)	2(1.2)	3(1.0)
	Unexpected worsening of
	other pre-existing condition	1(0.7)	0(0.0)	1(0.3)
	Lost to follow-up	5(3.4)	4(2.4)	9(2.9)
	Consent withdrawn
	(not due to adverse event)	0(0.0)	1(0.6)	1(0.3)
	Other adverse events*	3(2.0)	2(1.2)	5(1.6)
	Other reasons	1(0.7)	2(1.2)	3(1.0)
	_____________________________________________________________________
	* Tiotropium: cardio-respiratory arrest, chest pain, dry mouth, sudden death
	Placebo: rash, dry mouth, tremor, nausea and vomiting, constipation, weakness

Table 3

 3 Trough FEV 1 and FVC response (tiotropium -placebo) for change from baseline values at 12 weeks by smoking status

			FEV 1			FVC
		Difference,	95% CI	P-value	Difference,	95% CI	P-value
		LSM (ml)			LSM (ml)
	All patients	102	41, 164	0.0011	164	58, 270	0.0024
	Smokers	138	32, 244	0.0105	158	23, 341	0.0870
	Ex-smokers	66	3, 129	0.0375	170	62, 278	0.0021
	LSM, least square means; FEV 1 , forced expiratory volume in 1 second; FVC, forced
	vital capacity. *Differences between smoking groups were not significant (P=0.6982 for
	FEV 1 and P=0.5220 for FVC)

Table 4

 4 Adverse event profile

	Tiotropium	Placebo

Acknowledgements

The authors wish to acknowledge the following investigators for their contributions to the study: Dr Maria de La Salete Valente; Dr Ricardo Nascimento; Dr Carlos Boavida; Dr Ulisses Brito; Dr Conceição Antunes; Dr João Roque Dias; Dr Dias Pereira; Dr Teresa Cardoso; Dr Reis Ferreira; Dr Jorge Roldão Viera; Dr Abílio Reis; Dr Aida Coelho; Dr Sousa Barros; Dr Carlos Alves; Dr Amaral Marques PhD; Dr Paula Simão; Dr José Vieira; Dr Bugalho de Almeida PhD; Dr Olga Freitas; Dr Paula Duarte; Dr João de Almeida; Dr Mariano Machado; Dr Luís Goes; Dr Simões Torres; Dr Maria Manuel Machado; Dr Júlio Gomes; Dr Luís Oliveira. The authors also wish to acknowledge Dr José Antunes, Dr Ana Cristina Bastos, Esmeralda Violas and Conceição Peralta for their work in support of this study and PAREXEL MMS for editorial support in the preparation of this manuscript. Dr Joaquim Moita, Dr Cristina Bárbara PhD, Dr João Cardoso and Dr Rui Costa were the top recruiters of this study. Dr Marisa Sousa and Dr Jorge Ruiz are employees of Boehringer Ingelheim, and Dr Maria Luisa Santos participated in the trial as a clinical trials monitor at Pfizer. This study was funded by Boehringer Ingelheim and Pfizer.

A c c e p t e d m a n u s c r i p t


Ute Oltmanns

Matt Walters

Maria Sukkar

Sherry Xie

Razao Issa

Jane Mitchell

Malcolm Johnson

Kian Fan Chung

Professor Fan Chung
email: f.chung@imperial.ac.uk

Fluticasone, but not salmeterol, reduces cigarette smoke-induced production of interleukin-8 in human airway smooth muscle

Keywords: airway smooth muscle, chronic obstructive pulmonary disease, cigarette smoke, interleukin-8, glucocorticosteroids, β 2 -agonists

Cigarette smoke is the leading risk factor for the development of chronic obstructive pulmonary disease. We have recently shown that cigarette smoke extract synergises with TNFα in the induction of IL-8 from human airway smooth muscle cells. We have investigated the effect of fluticasone propionate, a corticosteroid, and salmeterol, a β 2 -adrenergic receptor agonist, on cigarette smoke extract-induced IL-8 production by human airway smooth muscle cells.

Human airway smooth muscle cells in primary culture were exposed to cigarette smoke extract and/or TNFα. (1 ng ml -1 ) with and without pretreatment with fluticasone (10 -13 -10 -8 M) and/or salmeterol (10 -11 -10 -6 M). IL-8 was analysed by ELISA. Fluticasone dose-dependently inhibited IL-8 release induced by cigarette smoke extract, TNFα or combined cigarette smoke extract and TNFα. However, while IL-8 release in the presence of cigarette smoke extract alone was completely inhibited by fluticasone, IL-8 production induced by cigarette smoke extract and TNFα was only partially reduced. Salmeterol alone had no effect on cigarette smoke extract and/or TNFα-induced IL-8 production from human airway smooth muscle cells. Combined fluticasone and salmeterol did not cause further inhibitory effects compared to fluticasone alone. Fluticasone but not salmeterol is effective in reducing cigarette smoke extract-induced IL-8 production in human airway smooth muscle cells. The reduced inhibition of cigarette smoke extract-and TNFα-induced IL-8 release by fluticasone may explain why corticosteroids are less effective in chronic obstructive pulmonary disease where increased amounts of TNFα are present.

A c c e p t e d m a n u s c r i p t Introduction

Chronic obstructive pulmonary disease is a major public health problem that is currently ranking as the fourth leading cause of death in the world [START_REF] Pauwels | 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[END_REF]. It is characterised by progressive and largely irreversible airflow limitation associated with symptoms such as cough, sputum production, and dyspnoea, and by a chronic inflammatory response which may represent a response to noxious particles and gases present in tobacco smoke. Thus, cigarette smoke represents a powerful inflammatory insult to the lungs which may be the most important pathological mechanism leading to chronic obstructive pulmonary disease. Increased numbers of inflammatory cells are found even in the lungs of asymptomatic smokers [START_REF] Niewoehner | Pathologic changes in the peripheral airways of young cigarette smokers[END_REF][START_REF] Mullen | Reassessment of inflammation of airways in chronic bronchitis[END_REF]. Cigarette smoke activates lung macrophages as well as resident lung cells such as epithelial cells and fibroblasts to release inflammatory mediators including tumour necrosis factor (TNF-) α and the neutrophil chemokine, interleukin (IL-) 8 [START_REF] Numanami | Serine protease inhibitors modulate smokeinduced chemokine release from human lung fibroblasts[END_REF][START_REF] Mio | Cigarette smoke induces interleukin-8 release from human bronchial epithelial cells[END_REF][START_REF] Walters | Cigarette smoke activates human monocytes by an oxidant-ap-1 signaling pathway: Implications for steroid resistance[END_REF]. These mediators, together with proteases and oxidants produced by activated neutrophils and macrophages, are capable of sustaining inflammation and damaging lung architecture [START_REF] Barnes | Chronic obstructive pulmonary disease: Molecular and cellular mechanisms[END_REF].

Human airway smooth muscle cells represent an important structural component of the airway wall. In addition to their traditionally accepted role as contractile cells, human airway smooth muscle cells are a rich source of chemokines including IL-8, eotaxin and RANTES, [START_REF] John | Induction of eotaxin expression and release from human airway smooth muscle cells by il-1beta and tnfalpha: Effects of il-10 and corticosteroids[END_REF]9]. Chemokines play an integral role in the coordination and persistence of airway inflammation by enhancing leukocyte migration into inflamed airways. Cytokines such as IL-1β, TNFα, IL-17 and transforming growth factor (TGF) β are capable of activating airway smooth muscle synthetic capacity [START_REF] John | Expression and release of interleukin-8 by human airway smooth muscle cells: Inhibition by th-2 cytokines and corticosteroids[END_REF][START_REF] Fong | Tgf-beta1 stimulates il-8 release, cox-2 expression, and pge(2) release in human airway smooth muscle cells[END_REF][START_REF] Rahman | IL-17R activation of human airway smooth muscle cells induces CXCL-8 production via a transcriptional-dependent mechanism[END_REF]. We recently found that cigarette smoke extracts activate human airway smooth muscle cells to release IL-8, an effect that was A c c e p t e d m a n u s c r i p t 5 significantly enhanced when cigarette smoke extract was combined with TNFα [START_REF] Oltmanns | Cigarette smoke induces il-8, but inhibits eotaxin and rantes release from airway smooth muscle[END_REF] and that a similar phenomenon occurs in human monocytes and macrophages [START_REF] Walters | Cigarette smoke activates human monocytes by an oxidant-ap-1 signaling pathway: Implications for steroid resistance[END_REF]. In contrast, the TNFα-induced production of eotaxin and RANTES by human airway smooth muscle cells was inhibited by cigarette smoke. The synergy between cigarette smoke and TNFα in the induction of IL-8 release from human airway smooth muscle cells is of clinical significance because inflammatory cytokines, such as TNFα, are present in the lungs of chronic obstructive pulmonary disease patients [START_REF] Kuschner | Dose-dependent cigarette smoking-related inflammatory responses in healthy adults[END_REF].

Currently, the established pharmacotherapy of chronic obstructive pulmonary disease consists of inhaled bronchodilators, including β 2 -agonists and anticholinergics and corticosteroids [START_REF] Pauwels | 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[END_REF]. The reported beneficial effects of inhaled corticosteroids in the treatment of chronic obstructive pulmonary disease have been somewhat variable.

Although recent studies indicate that regular treatment with inhaled corticosteroids does not influence the rate of decline of airflow obstruction, other studies have shown a reduction in the rate of exacerbations particularly in severe patients and a significant improvement in health status [START_REF] Burge | Randomised, double blind, placebo controlled study of fluticasone propionate in A c c e p t e d m a n u s c r i p t 18 patients with moderate to severe chronic obstructive pulmonary disease: The isolde trial[END_REF]. More effective symptom control and reduction in exacerbation rates is achieved by a combination of inhaled corticosteroids and longacting β 2 -agonist treatment [START_REF] Calverley | Combined salmeterol and fluticasone in the treatment of chronic obstructive pulmonary disease: A randomised controlled trial[END_REF][START_REF] Calverley | Maintenance therapy with budesonide and formoterol in chronic obstructive pulmonary disease[END_REF]. In addition, patients on combined fluticasone propionate and salmeterol therapy show a survival advantage over patients using each of these drugs alone [START_REF] Soriano | Inhaled corticosteroids with/without long-acting beta-agonists reduce the risk of rehospitalization and death in copd patients[END_REF] and current guidelines recommend treatment with both drugs for patients with moderate to severe chronic obstructive pulmonary disease [START_REF] Pauwels | 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[END_REF].

Previous studies have reported that there may be additive or synergistic effects of the combination of corticosteroids and β 2 -agonists on human airway smooth muscle cells in the inhibition of the release of IL-8 by TNFα [START_REF] Pang | Synergistic inhibition by beta(2)-agonists and corticosteroids on tumor necrosis factor-alpha-induced interleukin-8 release from cultured human airway smooth-muscle cells[END_REF]. For better applicability of these data to chronic obstructive pulmonary disease, we examined the potential mechanism by which corticosteroids and the combination of corticosteroids

Methods

Materials

Cigarettes (Phillip Morris, UK) were bought from a London high street supermarket.

Tissue culture reagents were obtained from Sigma (Poole, UK). Cell culture plasticware was purchased from Falcon Labware (Becton Dickinson, Oxford, UK).

Recombinant human TNFα and matched antibody pairs for IL-8 enzyme-linked immunosorbent assays (ELISA) were purchased from R&D Systems (DuoSet, Abingdon, UK). All other chemical reagents were obtained from Sigma (Poole, UK).

Isolation and culture of human airway smooth muscle cells

Human airway smooth muscle was obtained from lobar or main bronchus from patients undergoing lung resection for carcinoma of the bronchus. This study was approved by the Ethics Committee and, informed and signed consent was obtained from each patient. The smooth muscle was dissected out under sterile conditions and placed in culture as previously described [START_REF] Belvisi | Induction of cyclo-oxygenase-2 by cytokines in human cultured airway smooth muscle cells: Novel inflammatory role of this cell type[END_REF]. Cells were maintained in Dulbecco´s modified Eagle´s medium (DMEM) containing 10% fetal calf serum supplemented with sodium pyruvate (1mM), L-glutamine (2mM), non-essential amino acids (1:100), penicillin (100 U ml -1 )/streptomycin (100 µg/ml) and amphotericin B (1.5 µg ml -1 ) in a humidified atmosphere at 37°C in air/CO 2 (95:5 % vol/vol). At confluence, human airway smooth muscle cell cultures exhibited a typical hill-and-valley appearance.

Cigarette Smoke Extract

In order to prepare cigarette smoke extract, four cigarettes (filters removed) were combusted through a modified 60 ml syringe apparatus and the smoke passed through

Cytokine assay

Cell supernatants were harvested 24 hours after stimulation and stored at -70°C until assayed for IL-8. Cytokine levels were determined by using specific sandwich enzyme-linked immunosorbent assays (ELISA) according to the manufacturers` instructions.

Data analysis

Data are presented as mean ± SEM. Data were compared using one-way analysis of variance (ANOVA) followed by Bonferroni`s t test post hoc to determine statistical differences. A p value <0.05 was considered significant. SigmaStat software (Jandel Scientific, Germany) was used for statistical analysis.

A c c e p t e d m a n u s c r i p t

9

Results

Effect of fluticasone propionate and salmeterol on cigarette smoke induced IL-8 release

As shown previously [START_REF] Oltmanns | Cigarette smoke induces il-8, but inhibits eotaxin and rantes release from airway smooth muscle[END_REF], cigarette smoke extract (10%) and TNFα (1ng/ml) alone induced IL-8 release from human airway smooth muscle cells (41.1 ± 10.9 pg/ml and 400.12 ± 80.7 pg/ml, respectively), an effect that was significantly enhanced when cigarette smoke extract and TNFα were combined (743.12 ± 66.0 pg/ml). Fluticasone propionate inhibited cigarette smoke extract-induced IL-8 release from human airway smooth muscle cells in a dose dependent manner (IC 50 (-log M):

11.2 ± 0.7) with significant effect at 10 -10 to 10 -8 M (Figure 1). Complete inhibition of IL-8 release from human airway smooth muscle cells by fluticasone propionate was observed at 10 -9 M.

Salmeterol alone at a concentration of 10 -11 -10 -6 M had no effect on cigarette smoke extract-induced IL-8 production from human airway smooth muscle cells (data not shown). There was no enhanced inhibitory effect of fluticasone propionate on cigarette smoke extract-induced IL-8 release when combined with salmeterol (Figure 1). alone causes some degree of steroid resistance, similar to cigarette smoke extract and IL-1β in monocytes [START_REF] Walters | Cigarette smoke activates human monocytes by an oxidant-ap-1 signaling pathway: Implications for steroid resistance[END_REF] that was not reversed by the addition of salmeterol.

Effect of fluticasone propionate and salmeterol on

Neutrophils may mediate many of the aspects of lung damage seen in chronic obstructive pulmonary disease because they release various substances such as proteases, cytokines and oxidants that have deleterious effects on the airways and lung parenchyma [START_REF] Sutherland | Airway inflammation in chronic obstructive pulmonary disease: Comparisons with asthma[END_REF][START_REF] Chung | Cytokines in chronic obstructive pulmonary disease[END_REF]. Elevated levels of IL-8, a potent neutrophil chemoattractant, were found in the bronchoalveolar lavage fluid of smokers compared to nonsmokers and expression of IL-8 in lungs of patients with chronic obstructive pulmonary disease increases with progression of disease [START_REF] Mio | Cigarette smoke induces interleukin-8 release from human bronchial epithelial cells[END_REF][START_REF] Ito | Decreased histone deacetylase activity in chronic obstructive pulmonary disease[END_REF]. Accumulation of neutrophils in the lungs of patients with chronic obstructive pulmonary disease may therefore be linked to increased amounts of IL-8 in the lungs of smokers and patients with chronic obstructive pulmonary disease. protein-1 (AP-1), which regulate expression of various inflammatory genes including IL-8, may be involved [START_REF] Macnee | Oxidants and antioxidants as therapeutic targets in chronic obstructive pulmonary disease[END_REF]. Corticosteroids, after forming a complex with its cytoplasmic receptor, directly suppress the transcription of pro-inflammatory genes by binding to the promoter-enhancer regions of target genes. In addition, corticosteroids reduce the inflammatory effects of activator protein-1 and nuclear factor-κB by binding with these transcription factors and increasing the expression of the nuclear factor-κB inhibitor, IκBα [START_REF] Adcock | Molecular mechanisms of glucocorticosteroid actions[END_REF]. Both direct suppression of gene transcription (by transactivation) and interaction with transcription factors (by transrepression) may Glucocorticosteroids achieve significant control of clinical symptoms in chronic obstructive pulmonary disease. However, corticosteroids are less efficient in reducing airway inflammation in chronic obstructive pulmonary disease than in asthma and the mechanisms of this reduced effectiveness in chronic obstructive pulmonary disease as compared to asthma is not known [START_REF] Adcock | Overview: Why are corticosteroids ineffective in copd?[END_REF]. Oxidative stress may decrease the efficacy of corticosteroids in chronic obstructive pulmonary disease by reducing glucocorticoid receptor nuclear translocation and glucocorticoid receptor α expression [START_REF] Adcock | Overview: Why are corticosteroids ineffective in copd?[END_REF]. In line with this hypothesis, a recent study reported impaired efficacy of inhaled and oral corticosteroids in smoking asthmatics compared to non-smoking asthmatics [START_REF] Chalmers | Influence of cigarette smoking on inhaled corticosteroid treatment in mild asthma[END_REF][START_REF] Chaudhuri | Cigarette smoking impairs the therapeutic response to oral corticosteroids in chronic asthma[END_REF]. Cigarette smoke extract may indeed induce oxidative stress in human airway smooth muscle cells and the release of IL-8 induced by cigarette smoke extract is blocked by the antioxidant, glutathione [START_REF] Oltmanns | Cigarette smoke induces il-8, but inhibits eotaxin and rantes release from airway smooth muscle[END_REF]. Culpitt et al [START_REF] Culpitt | Impaired inhibition by dexamethasone of cytokine release by alveolar macrophages from patients with chronic obstructive pulmonary disease[END_REF] showed that dexamethasone inhibited IL-8 production by alveolar macrophages from smokers without evidence of chronic obstructive pulmonary disease but failed to inhibit IL-8 release from alveolar macrophages from patients with chronic obstructive pulmonary disease. Interestingly, we observed potent inhibition of cigarette smoke extractinduced IL-8 release by fluticasone propionate while the presence of TNFα significantly reduced the inhibitory effect of fluticasone propionate. Therefore, TNFα may induce resistance to the anti-inflammatory actions of corticosteroids and may contribute to steroid resistance in chronic obstructive pulmonary disease where increased amounts of TNFα are present [START_REF] Keatings | Differences in interleukin-8 and tumor necrosis factor-alpha in induced sputum from patients with chronic 32[END_REF]. Activation of mitogen-activated protein (MAP) kinases, such as p38 and c-Jun N-terminal kinase (JNK), may be linked to TNFα-induced steroid resistance. Mitogen-activated protein kinases, are important A which in turn leads to phosphorylation of the transcription factor, cAMP response element binding element. Interaction of cAMP response element binding element with its binding protein may then modulate gene transcription [START_REF] Shore | Regulation of beta-adrenergic responses in airway smooth muscle[END_REF]. In human airway smooth muscle cells, induction as well as suppression of gene expression by β 2agonists has been reported [START_REF] Shore | Regulation of beta-adrenergic responses in airway smooth muscle[END_REF]. Pang et al [START_REF] Soriano | Inhaled corticosteroids with/without long-acting beta-agonists reduce the risk of rehospitalization and death in copd patients[END_REF] reported induction of IL-8 release by salmeterol at a concentration of 10 -7 M with further increase at 10 -6 M; however, they saw no effect of salmeterol on TNFα-induced IL-8 release. In line with clinical trials demonstrating additive effects of combined corticosteroid and β 2 -agonist treatment [START_REF] Myo | Additive blockade of beta 2-integrin adhesion of eosinophils by salmeterol and fluticasone propionate[END_REF], combination of salmeterol and fluticasone propionate enhanced inhibition of IL-8 release from human airway smooth muscle cells compared to fluticasone propionate alone [START_REF] Soriano | Inhaled corticosteroids with/without long-acting beta-agonists reduce the risk of rehospitalization and death in copd patients[END_REF]. In a study by Ammit et al, TNFα-induced RANTES release from human airway smooth muscle cells was inhibited by dexamethasone with further additive inhibition when salmeterol was added to dexamethasone [START_REF] Ammit | Tumor necrosis factor-alpha-induced secretion of rantes and interleukin-6 from human airway smooth muscle cells: Modulation by glucocorticoids and beta-agonists[END_REF]. We saw no effect of salmeterol alone at 10 -11 -10 propionate was not enhanced when combined with salmeterol. We obtained similar data when examining effects on combined cigarette smoke extract and TNFα effects.

We have shown that fluticasone propionate but not salmeterol is effective in reducing smoking-induced IL-8 release from human airway smooth muscle cells. The increased presence of pro-inflammatory cytokines such as TNFα in airway inflammation in chronic obstructive pulmonary disease may impair the antiinflammatory actions of corticosteroids in chronic obstructive pulmonary disease.

Grant support

This study was supported by a grant from the Wellcome Trust and from GSK R&D, UK.

Competing interests

KFC has been renumerated for participation at Advisory Board meetings of GSK, Astra-Zeneca and Novartis, and has received unrestricted research grant from GSK.

MJ is an employee of GSK. Other authors have no competing interests.

A c c e p t e d m a n u s c r i p t 6 and

 6 β2-agonists may be beneficial in patients with chronic obstructive pulmonary disease by studying whether fluticasone propionate and salmeterol alone or in combination can modulate cigarette smoke extract-induced IL-8 release.

A c c e p t e d m a n u s c r i p t 7 Immunofluorescence

 7 techniques for calponin, smooth muscle α-actin and myosin heavy chain revealed that more than 95% of the cells displayed the characteristics of smooth muscle cells in culture. Human airway smooth muscle cells at passages 3-7 from 8 different donors were used.

A c c e p t e d m a n u s c r i p t 8 (

 8 100 mls of Dulbecco´s modified Eagle´s medium. Each cigarette yielded 5 draws of 60 ml of the syringe, with each individual draw taking approximately 10 seconds to complete. This solution represents '100%' strength. Smoked medium was then passed through a 0.25 µm filter in order to sterilise the solution. Smoked medium was diluted to the required strength in Dulbecco´s modified Eagle´s medium and placed upon the cells immediately afterwards.Cell treatmentPrior to the experiments, confluent cells were growth-arrested by fetal calf serum deprivation for 24h in Dulbecco´s modified Eagle´s medium supplemented with sodium pyruvate (1 mM), L-glutamine (2 mM), non-essential amino acids (1:100), penicillin (100 U/ml)/ streptomycin (100 µg/ml), amphotericin B (1.5 µg/ml), insulin (1 µM), transferrin (5 µg/ml), ascorbic acid (100 µM) and bovine serum albumin (0,1 %). Cells were then exposed to cigarette smoke extract (10 %) in the presence and absence of TNFα (1 ng/ml). In additional experiments cells were pretreated with fluticasone propionate (10 -13 -10 -8 M) and/or salmeterol 10 -11 -10 -6 M) for 2 hours before adding cigarette smoke extract or cigarette smoke extract and TNFα.

A c c e p t e d m a n u s c r i p t 10 completeA c c e p t e d m a n u s c r i p t 11 Discussion

 1011 TNFα and cigarette smoke induced IL-8 release IL-8 release from human airway smooth muscle cells induced by either TNFα alone or by the combination of TNFα and cigarette smoke extract, was dose-dependently inhibited by fluticasone propionate (IC 50 (-log M): 10.7 ± 0.6; 10.7 ± 0.8, respectively) with maximum inhibition (65.3 ± 3.2% and 75.5 ± 4.3%, respectively) observed at the highest concentration used (10 -8 M, Figure 2 and 3). In contrast to inhibition of cigarette smoke extract-induced IL-8 release by fluticasone propionate at 10 -9 M, IL-8 production in the presence of TNFα and cigarette smoke extract was reduced by 62.1 ± 3.5% (p<0.001), associated with a right-shift of the concentration-response curve (Figure 4). The combination of fluticasone propionate and salmeterol showed no enhanced inhibitory effect compared to fluticasone propionate alone. We recently showed that cigarette smoke extract induces IL-8 release from human airway smooth muscle cells and synergises with TNFα in enhancing IL-8 production [13]. In the present study, fluticasone propionate inhibited IL-8 release induced by cigarette smoke extract, TNFα or the combination of cigarette smoke extract and TNFα in a dose-dependent manner. However, while IL-8 release in the presence of cigarette smoke extract alone was abolished by fluticasone propionate at 10 -9 M, IL-8 production induced by cigarette smoke extract and TNFα was reduced by 62.1 ± 3.5% only, indicating reduced efficacy of fluticasone propionate in the presence of TNFα. In contrast, salmeterol had no effect on cigarette smoke extract and/or TNFα induced IL-8 production from human airway smooth muscle cells. The combination of fluticasone propionate and salmeterol did not result in enhanced inhibitory effect compared to fluticasone propionate alone. Our data show that TNFα

A c c e p t e d m a n u s c r i p t 12 Glucocorticosteroids

 12 and long-acting β2-agonists remain central to the management of patients with chronic obstructive pulmonary disease[START_REF] Calverley | Combined salmeterol and fluticasone in the treatment of chronic obstructive pulmonary disease: A randomised controlled trial[END_REF]. Human airway smooth muscle cells express both β 2 -adrenergic and glucocorticoid receptors and therefore represent interesting target cells for treatment with corticosteroids and β 2 -agonists in inflammatory lung diseases. Previous studies have shown that corticosteroids are effective in inhibiting cytokine release from human airway smooth muscle cells[START_REF] John | Expression and release of interleukin-8 by human airway smooth muscle cells: Inhibition by th-2 cytokines and corticosteroids[END_REF][START_REF] John | Inhibition of chemokine production from human airway smooth muscle cells by fluticasone, budesonide and beclomethasone[END_REF]. Dose-dependent inhibition of TNFα-induced IL-8 release by fluticasone propionate (10 -8 -10 -6 M) has been shown previously[START_REF] Pang | Synergistic inhibition by beta(2)-agonists and corticosteroids on tumor necrosis factor-alpha-induced interleukin-8 release from cultured human airway smooth-muscle cells[END_REF]. In the present study, we used a wider range of concentrations to investigate the effect of fluticasone propionate on TNFα-induced IL-8 release and show that significant inhibitory effects occurred at a fluticasone propionate concentration as low as 10 -10 M. In addition, fluticasone propionate was very potent in inhibiting cigarette smoke extract-induced IL-8 production from human airway smooth muscle cells with complete inhibition at concentrations as low as 10 -9 M. The mechanisms involved in the cigarette smoke extract-induced cytokine release are not clear. However, oxidative stress-mediated activation of transcription factors such as nuclear factor-κB (NF-κB) and activator

A c c e p t e d m a n u s c r i p t 13 therefore

 13 contribute to inhibition of cigarette smoke extract-induced IL-8 release by fluticasone propionate.

A c c e p t e d m a n u s c r i p t 14 mediators

 14 of TNFα-induced gene activation in human airway smooth muscle cells[32]. Szatmary et al[START_REF] Szatmary | Inhibition of glucocorticoid receptormediated transcriptional activation by p38 mitogen-activated protein (map) kinase[END_REF] showed suppression of GR function by activated JNK and p38 MAP kinase indicating a potential mechanism behind reduced efficacy of steroids in chronic obstructive pulmonary disease. Activation of p38 MAP kinase has also been reported as underlying the reduced affinity of glucocorticoid receptor for its ligand induced by combination of IL-2 and IL-4 in peripheral blood mononuclear cells[START_REF] Irusen | P38 mitogen-activated protein kinase-induced glucocorticoid receptor phosphorylation reduces its activity: Role in steroid-insensitive asthma[END_REF].

β 2 -

 2 agonists bind to the G-protein-coupled seven-transmembrane β 2 -adrenoreceptor to activate adenylyl cyclase resulting in the formation of intracellular cyclic adenosine monophosphate (cAMP). cAMP activates protein kinase

A c c e p t e d m a n u s c r i p t 15 period.

 15 In addition, inhibition of TNFα-induced IL-8 release by fluticasone

A c c e p t e d m a n u s c r i p t 20 obstructiveA c c e p t e d m a n u s c r i p t 21

 2021 pulmonary disease or asthma. Am J Respir Crit Care Med. 1996; 153:530-534.

Figure 1 :

 1 Figure 1: Effect of increasing concentrations of fluticasone propionate (10 -13 -10 -8 M;) alone and in combination with salmeterol (10 -7 M; ) on cigarette smoke-induced

Figure 2 :

 2 Figure 2: Effect of increasing concentrations of fluticasone propionate (10 -13 -10 -8 M;) alone and in combination with salmeterol (10 -7 M; ) on TNFα-induced IL-8

Figure 3 :

 3 Figure 3: Effect of increasing concentrations of fluticasone propionate (10 -13 -10 -8 M;) alone and in combination with salmeterol (10 -7 M; ) on IL-8 release from human

Figure 4 :A c c e p t e d m a n u s c r i p t 22 smokeA c c e p t e d m a n u s c r i p t 23 Figure 1 A c c e p t e d m a n u s c r i p t 24 Figure 2 A c c e p t e d m a n u s c r i p t 25 Figure 3 A c c e p t e d m a n u s c r i p t 26 Figure 4

 422231242253264 Figure 4: Comparison of the effect of increasing concentrations of fluticasone propionate (10 -13 -10 -8 M) on IL-8 release from human airway smooth muscle cells in