Asthma Prosurvival activity for airway neutrophils in Thorax: first published as 10.1136/thx.2009.120741 on 4 August 2010. Downloaded from severe asthma Mohib Uddin,1 Guangmin Nong,1 Jonathan Ward,1 Gre´gory Seumois,1 Lynne R Prince,2 Susan J Wilson,1Victoria Cornelius,1 Gordon Dent,1 Ratko Djukanovic1 See Editorial, p 665 ABSTRACT of patients with severe asthma, a clear mechanistic < Supplementary methods and Background Airway neutrophilia is a recognised feature association between these mediators and airways a figure are published online of chronic severe asthma, but the mechanisms that neutrophilia has not yet been demonstrated. only. To view these files please underlie this phenomenon are unknown. Evidence for Theoretically, the observed neutrophilia in the visit the journal online (http:// factors present in airway secretions that prolong airways of those with asthma could be due to thorax.bmj.com). neutrophil survival has been sought and it has been increased chemotactic activity produced in the 1 Southampton NIHR Respiratory hypothesised that these might be augmented in inflamed airways or longer survival due to locally Biomedical Research Unit, neutrophilic asthma. produced factors, such as TNFa and GM-CSF, Southampton Wellcome Trust 11 Clinical Research Facility, Methods Non-smoking subjects with severe asthma which delay their apoptosis. It is increasingly Division of Infection, (SA) or mild asthma (MA) and healthy control subjects recognised that diverse phenotypes exist in Inflammation and Immunity, (HC) underwent sputum induction. The SA group was asthma.12 13 It is also known that there is marked University of Southampton subdivided into subjects with neutrophil counts above variability in airway cytokine/chemokine/growth School of Medicine, fi Southampton General Hospital, (SA-high) and those within the normal range (SA-low). factor pro les, expression of receptors and signal- 414e17 Southampton, UK Apoptotic neutrophils were enumerated in the cellular ling pathways, all of which could represent 2Academic Unit of Respiratory phase while the fluid phase was assessed for its ability different phenotypes. In a subpopulation of Medicine, School of Medicine & to prolong the in vitro survival of blood-derived subjects with severe asthma, partial or complete Biomedical Sciences, University neutrophils using morphometric and flow cytometric 2 of Sheffield, Royal Hallamshire steroid resistance is seen. Paradoxically, cortico- Hospital, Sheffield, UK analyses. steroid treatment itself could contribute to airway Results There was a significant difference between all neutrophilia in poorly controlled asthma by e Correspondence to four subject groups with respect to the percentage of promoting neutrophil survival.18 20 While the Professor Ratko Djukanovic, apoptotic sputum neutrophils (KruskaleWallis, exact role of neutrophils in asthma is still Southampton NIHR Respiratory ¼ Biomedical Research Unit, p 0.042). Cuzick test showed a highly significant unknown, an extended lifespan and/or failed Division of Infection, (p¼0.008) trend towards decreasing numbers of clearance of apoptotic neutrophils could enhance Inflammation and Immunity, apoptotic neutrophils across the four groups with the proinflammatory potential of these cells to http://thorax.bmj.com/ School of Medicine, University increasing asthma severity and neutrophil count. The cause lung tissue damage and to impede the of Southampton School of sputum antiapoptotic activity was also different between resolution of asthmatic inflammation.21 Medicine, Mailpoint 810, Level ¼ F, Sir Henry Wellcome the groups (p 0.039), with a highly significant In order to elucidate the mechanisms that lead to Laboratories, South Block, (p¼0.005) decreasing trend across the four groups. The airways neutrophilia in severe asthma, we have Southampton General Hospital, survival effect could not be inhibited by blocking selective conducted a cross-sectional study of subjects with Southampton SO16 6YD, UK; chemotaxin receptors, neutralising neutrophil survival mild asthma treated with b -agonists alone and [email protected]. 2 uk factors, inhibiting phosphatidylinositol-3-kinase (using those with severe asthma on high dose inhaled LY294002) or with pertussis toxin pretreatment. (ICS) and oral corticosteroids (OCS), using healthy on September 30, 2021 by guest. Protected copyright. MU and GN contributed equally Similarly, it could not be explained by lipopolysaccharide individuals as control subjects. We have hypoth- to this work. contamination or by the presence of inhaled esised that raised neutrophil numbers in more corticosteroids in sputum. severe asthma are the result of their prolonged Received 13 July 2009 fl Accepted 1 June 2010 Conclusions These data demonstrate the capacity of as survival promoted by factors released by in am- yet unidentified factor(s) in the airways of subjects with matory and structural cells of the airways. In order asthma to delay human neutrophil apoptosis and extend to test this hypothesis, we first quantified the their lifespan as a potential mechanism contributing to extent of in vivo apoptosis by enumerating sputum unresolving airways neutrophilia in severe asthma. neutrophils that display characteristic morpholog- ical features of apoptosis. Further evidence of increased survival in asthmatic airways was then sought by studying whether factors present in the INTRODUCTION fluid phase of sputum, which samples the epithelial Accumulating evidence points to an important role lining fluid (ELF), could alter the proportions of for neutrophils during acute exacerbations of neutrophils that become apoptotic in neutrophil e asthma and in severe forms of the disease1 3 where cultures. Any effect of residual corticosteroids in raised neutrophil numbers have been observed in sputum samples was tested by pretreating neutro- bronchoalveolar lavage (BAL), induced sputum and phils with the glucocorticoid (GC) receptor antag- both bronchial and transbronchial biopsies.145 onist, mifepristone (RU486), added to neutrophil While increased concentrations of neutrophil-active survival assays.22 The mechanisms underlying any 67 fl mediators interleukin 8 (IL-8), leukotriene B4 proneutrophil survival effect of the sputum uid 78 e (LTB4), granulocyte macrophage colony-stimu- phase were then studied using selective blockers for lating factor (GM-CSF)9 and tumour necrosis factor mediator pathways known to delay neutrophil a (TNFa)10 have been detected in BAL and plasma apoptosis. 684 Thorax 2010;65:684e689. doi:10.1136/thx.2009.120741 Asthma METHODS subsequent inhibition experiments, in which inhibitors of Thorax: first published as 10.1136/thx.2009.120741 on 4 August 2010. Downloaded from Subjects various mediators and signalling pathways were used to block Non-smoking volunteers, classified as severe atopic asthmatics the effect of sputum on in vitro apoptosis, the extent of (SA; n¼15), mild atopic asthmatics (MA; n¼11) or healthy apoptosis was assessed by flow cytometry using fluorescein control subjects (HC (n¼12) using established GINA (Global isothiocyanate (FITC)-labelled Annexin V (BD Biosciences, Initiative for Asthma) guidelines, were enrolled. The SA subjects Oxford, UK) and a FACSCalibur with Cell Quest software (BD were further divided into two subgroups according to the Biosciences) by counting 10 000 events per sample. We have ¼ fi percentage of neutrophils in sputum using the 65% cut-off for previously shown a strong (rs 0.70) and highly signi cant normal neutrophil counts reported by Belda and co-workers23 to (p<0.0001) correlation between the extent of neutrophil define SA with high counts (SA-high) and SA with low counts apoptosis measured using standard morphological criteria and (SA-low) (for criteria see the online supplement). The study was the extent quantified by flow cytometry.27 For further details of approved by the Southampton and South West Hampshire all the experimental methods see the online supplement. Ethics committee and all subjects were recruited using adver- tisements in the media, either directly or via a departmental Statistics database. All statistical analyses were performed using Prism version 5 for Windows (GraphPad Software, San Diego, California, USA) 28 Sputum induction and processing except the Cuzick test which was performed using STATA, Sputum induction was conducted using guidelines of the ERS Version 11.0 (StataCorp, College Station, Texas, USA). Sputum (European Respiratory Society) Task Force on induced sputum24 neutrophil numbers and neutrophil survival in in vitro assays with 4.5% hypertonic saline as described previously.25 were not normally distributed. These data are therefore expressed as median, IQR and range from the indicated numbers of experiments. Differences in these indices among the four Sputum cell counts and quantification of apoptotic neutrophils groups of patients were assessed by the KruskaleWallis test. Immunocytochemistry was applied to sputum cell phase cyto- fi 26 Where this returned a signi cant difference, the Cuzick non- spins to identify neutrophils using mouse monoclonal anti- parametric test for trend across the four groups (HC, MA, SA- neutrophil elastase antibody, followed by secondary rabbit low and SA-high) was performed.28 The effects of drugs on the e antimouse bionylated immunoglobulin G (IgG), streptavidin AP antiapoptotic activity of sputum supernatants on neutrophils in (alkaline phosphatase) and Fast Red, and counterstained with vitro were assessed using the paired Wilcoxon signed-rank test. A fi haematoxylin.