Neutrophilic inflammation in severe asthma



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Pro-survival activity for airway neutrophils in severe asthma

Mohib Uddin,1 Guangmin Nong,1 Jonathan Ward,1 Grégory Seumois,1 Lynne R. Prince,2 Susan Wilson,1 Victoria Cornelius,1 Gordon Dent1 and Ratko Djukanović1

1 Southampton NIHR Respiratory Biomedical Research Unit, Southampton Wellcome Trust Clinical Research Facility, Division of Infection, Inflammation and Immunity, University of Southampton School of Medicine, Mailpoint 810, Level F, South Block, Southampton General Hospital, Southampton SO16 6YD, United Kingdom

2Academic Unit of Respiratory Medicine, School of Medicine & Biomedical Sciences, University of Sheffield, L Floor, Royal Hallamshire Hospital, Sheffield S10 2JF, United Kingdom.

Correspondence and reprint requests to:

Professor Ratko Djukanović, Southampton NIHR Respiratory Biomedical Research Unit, Division of Infection, Inflammation and Immunity, School of Medicine, University of Southampton School of Medicine, Mailpoint 810, Level F, Sir Henry Wellcome Laboratories, South Block, Southampton General Hospital, Southampton SO16 6YD, United Kingdom.

E-mail: R.Djukanovic@southampton.ac.uk

Methods Supplementary Information

Subjects

Non-smoking volunteers comprising of 15 severe atopic asthmatics, 11 mild atopic asthmatics and 12 healthy non-atopic control subjects were recruited (see Table 1). Subjects were classified as mild if they had symptoms >2x/week but less than daily, if night-time symptoms occurred 80% of predicted values. They were classified as severe if they had daytime symptoms and used the bronchodilator salbutamol daily, had nocturnal symptoms >2x/month, and had FEV1800 μg per day) and a long-acting inhaled β2-agonist. Eleven of 15 were also on OCS. The control subjects had no history of smoking or respiratory symptoms and all had FEV1 >90% of predicted and were nonatopic. All the severe asthmatic subjects had been free of respiratory infection within the previous 4 weeks and had negative sputum culture (see Supplementary Table S1).

The study was approved by the Southampton and South West Hampshire Ethics committee and all subjects were recruited using advertisements in the media, either directly or via a departmental database.

Sputum induction and processing

Sputum induction was conducted using guidelines of European Respiratory Society’s Task Force on induced sputum (S1) and as described previously (S2). The mucoid portions were split into two parts and mixed with either four volumes of phosphate-buffered saline (PBS) or equal volumes of dithioerythritol (DTE). Both suspensions were filtered and centrifuged to separate the cell and fluid phases. The DTE-processed samples were used to generate cytospins for cell counts while the PBS-processed samples were used for the neutrophil apoptosis assays.

Sputum cell counts and quantification of apoptotic neutrophils

Immunocytochemistry was used for accurate identification of apoptotic neutrophils (S3) using mouse monoclonal anti-neutrophil elastase antibody (1:1000 dilution) followed by further incubation with rabbit anti-mouse bionylated IgG (1:200 dilution). Thereafter, slides were incubated with Streptavidin-AP and Fast Red and slides counterstained with haematoxylin. The immunostained cells were identified as apoptotic based on typical morphological features of neutrophil apoptosis: cell shrinking, nuclear condensation and fragmentation, plasma membrane ruffling and blebbing (S4).

In vitro assessment of anti-apoptotic activity of sputum

The effect of sputum on neutrophil survival in vitro was assessed by culturing blood-derived neutrophils for 22 hr in the presence or absence of PBS-processed fluid phase of sputum (1:20 dilution). LPS (10 ng/ml), known to enhance neutrophil survival, was used as a positive control. In order to avoid growth of any bacteria in sputum all supernatants were irradiated (10 Gray) before culture.

For the apoptosis assays, neutrophils were obtained from normal healthy donors and purified as previously described (S5) and resuspended in RPMI 1640 with 20% fetal calf serum (FCS) plus 2% L-glutamine, sodium pyruvate supplemented with 50 U/ml streptomycin and penicillin at 37° C in a humidified atmosphere of 5% CO2 (v/v) in air. After incubation for 22 hr, neutrophils were harvested by centrifugation at 300 g for 10 min at 4oC, washed twice and resuspended with 10% heating-inactivated FCS/PBS. In the first series of experiments, the numbers of apoptotic cells were quantified using cytocentrifuged cell preparations fixed in methanol and stained with Diff-QuikTM. Morphometric assessment of apoptosis was performed microscopically (x 100 objective oil immersion light microscopy) by counting at least 400 cells/slide with the observer blinded to the assay conditions and numbers of apoptotic cells expressed as a percentage of total cell counts. This method has been widely used in studies elucidating mechanisms of neutrophil apoptosis (S4).

In preliminary 5 experiments, mifepristone (1 µM) was added to block any effects of ICS or OCS present in sputum, and dexamethasone (100 nM) was used as control. Dexamethasone and mifepristone were dissolved in ethanol (EtOH) with a final vehicle concentration in culture as 0.01% v/v which was found not to affect constitutive neutrophil apoptosis (data not shown). In all instances, dexamethasone prolonged neutrophil survival and this was completely abolished by mifepristone (data not shown). The concentrations of sputum supernatant, dexamethasone and mifepristone were chosen on the basis of preliminary dose–response experiments (data not shown).

In subsequent experiments in which inhibitors against various mediators and signalling pathways were used to block the effect of sputum on in vitro apoptosis, the extent of apoptosis was assessed by flow cytometry using FITC-labeled Annexin V (BD Biosciences). Neutrophils (106) were removed from culture (22 hr) and washed in PBS then resuspended in 50 μL FITC-labeled Annexin V (1 μg/ml) and propidium iodide (12.5 μg/ml) in Annexin V binding buffer (10 mM HEPES, pH 7.4, 140 mM NaCl, 2.5 mM CaCl2). Samples were incubated for 15 min at 4°C in the dark, and then diluted with 100 µl of binding buffer prior to analysis by flow cytometry using a FACSCalibur with Cell Quest software (BD Biosciences, Oxford, U.K) by counting 10,000 events per sample.

Figure S1. Morphometric assessment of neutrophil apoptosis using light microscopy.

(A) Normal neutrophils characterised by their multi-lobular nuclei. (B) Apoptotic neutrophils displaying loss of chromatin bridges (white arrow) and condensed nucleus (black arrow).

A B

[pic]

References

S1. Paggiaro PL, Chanez P, Holz O, Ind PW, Djukanovic R, Maestrelli P, Sterk PJ. Sputum induction. Eur Respir J 2002;37:3s-8s.

S2. Yoshikawa T, Dent G, Ward J, Angco G, Nong G, Nomura N, Hirata K, Djukanovic R. Impaired neutrophil chemotaxis in chronic obstructive pulmonary disease. Am J Respir Crit Care Med 2007;175:473-479.

S3. Pulford KA, Erber WN, Crick JA, Olsson I, Micklem KJ, Gatter KC, Mason DY. Use of monoclonal antibody against human neutrophil elastase in normal and leukaemic myeloid cells. J Clin Path 1988;41:853-860.

S4. Haslett C. Granulocyte apoptosis and inflammatory disease. Br Med Bull 1997; 53:669-683.

S5. Rytila P, Plataki M, Bucchieri F, Uddin M, Nong G, Kinnula VL, Djukanovic R. Airway neutrophilia in COPD is not associated with increased neutrophil survival. Eur Respir J 2006;28:1163-1169.

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