Reduced glucocorticoid receptor expression and function in airway neutrophils

International Immunopharmacology 12 (2012) 26–33

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Reduced glucocorticoid receptor expression and function in airway neutrophils Jonathan Plumb a,⁎, Kate Gaffey a, Binita Kane a, Brendan Malia-Milanes a, Rajesh Shah a, Andrew Bentley a, David Ray b, Dave Singh a a b

University of Manchester, NIHR Translational Research Facility, Manchester Academic Health Science Centre, University Hospital of South Manchester, Manchester, UK Centre for Molecular Medicine, University of Manchester, Manchester Academic Health Science Centre, England, UK

a r t i c l e

i n f o

Article history: Received 25 July 2011 Received in revised form 8 September 2011 Accepted 6 October 2011 Available online 25 October 2011 Keywords: COPD Glucocorticoid receptor Neutrophil

a b s t r a c t Chronic Obstructive Pulmonary Disease (COPD) is a glucocorticoid resistant condition characterised by airway neutrophilia. Reduced glucocorticoid receptor (GR) expression in COPD airway neutrophils may be a mechanism that contributes to glucocorticoid resistance. Our objective was to investigate the expression and function of GR within COPD airway neutrophils. Dual-label immunofluorescence was used to analyse airway neutrophil expression of GR within peripheral lung tissue samples (11 COPD patients, 7 healthy non-smokers [NS]) and induced sputum (7 COPD patients, 7 NS). TNFα and CXCL8 release were measured in neutrophils isolated from induced sputum and peripheral blood (7 COPD patients) in the presence of dexamethasone. In lung tissue, GR was abundantly expressed in macrophages and lymphocytes, but very low expression was observed in neutrophils (means 6.8% and 4.3% in COPD patients and NS respectively). Similarly low expression was observed in sputum neutrophils (means 3.8% and 6.9% in COPD patients and NS respectively). In contrast, GR was expressed by 100% of blood neutrophils. Dexamethasone had less suppressive effect on TNFα and CXCL8 production in vitro by neutrophils from induced sputum compared to neutrophils from paired blood samples. Airway neutrophils have low expression of GR in both COPD patients and controls. The effects of glucocorticoids on cytokine production from airway neutrophils are reduced. Increased numbers of airway neutrophils lacking GR may contribute to glucocorticoid resistance in COPD patients. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Chronic obstructive pulmonary disease (COPD) is characterised by progressive airway inflammation. There are increased numbers of neutrophils within the airway lumen [1,2] and airway wall [3,4] in COPD patients. Neutrophils are thought to play a key role in the pathophysiology of COPD as they secrete a wide range of pro-inflammatory cytokines and chemokines, as well as tissue destructive proteases [5]. Neutrophils are therefore a target for anti-inflammatory therapies in COPD [6]. Glucocorticoids are the most widely used anti-inflammatory treatment for COPD, but their clinical effectiveness is limited [7,8]. Glucocorticoids bind to the α isoform of the cytoplasmic glucocorticoid receptor (GR), forming a complex that inhibits the activity of ⁎ Corresponding author at: University of Manchester, NIHR Translational Research Facility, Manchester Academic Health Science Centre, University Hospital of South Manchester Foundation Trust, Southmoor Road, Manchester, M23 9LT, UK. Tel.: + 44 161 291 5920; fax: + 44 161 291 5806. E-mail addresses: [email protected] (J. Plumb), [email protected] (K. Gaffey), [email protected] (B. Kane), [email protected] (B. Malia-Milanes), [email protected] (R. Shah), [email protected] (A. Bentley), [email protected] (D. Ray), [email protected] (D. Singh). 1567-5769/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.intimp.2011.10.006

transcription factors such as NFκB, thereby reducing inflammatory gene expression [9]. Cellular glucocorticoid sensitivity is proportional to GRα expression [11]. GR is expressed in epithelial cells, smooth muscle cells, endothelial cells and infiltrating inflammatory cells in the lungs of both healthy subjects and COPD patients [10]. GR expression specifically in airway neutrophils in COPD is relatively unexplored. We have investigated the expression and function of GR in airway neutrophils from COPD patients. GR expression in airway neutrophils from COPD patients and controls was studied in the small airways using peripheral lung tissue, and in the more proximal airways using induced sputum samples. The effect of the glucocorticoid dexamethasone on cytokine production from airway neutrophils was studied using neutrophils cultured from induced sputum samples. 2. Methods 2.1. Subjects For lung immunohistochemistry, 11 COPD patients, defined using GOLD guidelines [12] and 7 lifelong non-smokers without airflow obstruction (NS) undergoing lobectomy for suspected or confirmed lung cancer were recruited. For induced sputum collection, 14 COPD

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patients and 7 NS were recruited. We excluded subjects with a history of asthma, respiratory tract infection within 4 weeks or presence of any significant concurrent medical conditions. Demographics for all of the participants are shown in Table 1. Subjects gave written informed consent. The study was approved by the local research ethics committee. 2.2. Induced sputum Sputum was induced and dithiothreitol (DTT) processed using established methods [13]. Sputum cell pellets were resuspended in RPMI 1640 supplemented with 10% foetal calf serum (FCS), 1% penicillin–streptomycin, and 1 mmol/L L-glutamine at a concentration of 1 × 106 cells/ml. 4 × 105 cells were incubated on a 24 well plate for 1 h at 37 °C in humidified 5% CO2. An enrichment procedure was performed using macrophage adherence to increase the proportion of neutrophils in culture. After 1 h the non-adherent cells (largely neutrophils) were removed and centrifuged at 2000 rpm for 10 min. The cells were then resuspended, and cell counts and cell viability were determined using the trypan blue exclusion method in a naubauer haemocytometer. 2.3. Isolation of blood neutrophils 4 ml of venous blood was layered over 3 ml of MonoPoly resolving medium (MP Biomedicals, Cambridge, UK) and centrifuged at 800 g for 45 min at 18 °C. Polymorphonuclear (PMN) leukocytes were removed and washed in RPMI 1460 (Sigma, Poole, UK). The cells were then resuspended, and cell counts and cell viability were determined using the trypan blue exclusion method in a naubauer haemocytometer. Cytopsins were prepared using PMNs at a concentration of 0.5 × 10 6/ml for differential cell counts. Cells were classified as eosinophils, lymphocytes, macrophages, and neutrophils based on morphology and staining and expressed as a percentage of total cell counts. 2.4. Immunofluorescence 2.4.1. Lung tissue Tissue blocks were taken, as far distal to tumour as possible, then formalin fixed and paraffin embedded. 4 μm sections were cut and lifted onto polysine coated glass slide (Surgipath, Peterborogh, UK). The cellular distribution of GR within the lung tissue was determined by immunofluorescence. GRM20 which identifies both GR α and β isoforms (Sc-1004, Santa Cruz Biotechnology, CA, USA) was duallabelled with the following phenotypic markers; T cells, (CD3, PS1, Novocastra, Newcastle, UK), macrophages (CD68, PG-M1, Dako, Ely, UK), Neutrophils (Neutrophil elastase (NE), NP57, Dako, Ely, UK) and epithelial cells (Cytokeratin, MNF116, Dako, Ely, UK). Sequential dual label immunofluorescence was carried out for GRM20 followed by CD3, CD68 and cytokeratin (CK). Optimal heat induced epitope retrieval (HIER) was carried out for GRM20, CD3, CD68

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and CK by microwaving sections in Tris-EDTA buffer pH9 (10 mM Tris Base, 1 mM EDTA, 0.5% Tween 20) for 20 min at 800 W. Sections to be stained for NE did not require HIER and were detected prior to the required HIER pre-treatment for GRM20. Primary antibodies were diluted in 1.5% normal serum (Vector Labs, Peterborough, UK) and applied overnight at 4 °C. Primary antibodies were detected using either Alexa 488 conjugated goat anti-rabbit IgG (for GR, 1:200, Invitrogen, Paisley, UK) or Alexa 568 conjugated goat anti-mouse IgG (for cell phenotypic markers). Sections were then incubated in 4′, 6diamidino-2-phenylindole (DAPI, Invitrogen, Paisley, UK) to act as a fluorescent nuclear counter stain. Omission of primary antibody on serial sections or incubation in suitable isotype control antibodies (Vector Labs, Peterborough, UK) were used as negative controls. On a subgroup of patients (n = 4) an anti GR mouse monoclonal antibody specific for GR α (Clone 41, 1:200, BD Biosciences, Oxford, UK) was also applied. 2.4.2. Cytospins Cytospins were created from sputum cells and peripheral blood neutrophils suspended in phosphate buffered saline at 1 × 10 6/ml. Cytospins were incubated in 0.1% sodium borohydride (Sigma, Poole, UK) in Tris-buffered saline for 10 min at room temperature to reduce autofluorescence. Cells were washed and then fixed for 5 min at −20 °C in ice-cold acetone:methanol (1:1 v:v), followed by blocking in normal serum. Cytospins were then incubated with the following primary antibodies overnight at 4 °C; GRM20 (Sc-1004, Santa Cruz Biotechnology, CA, USA) and neutrophil elastase (NE, NP57, Dako, Ely, UK). GR and NE were detected using Alexa 488 conjugated goat anti-rabbit IgG and Alexa 568 conjugated goat antimouse IgG (1:200, Invitrogen, Paisley, UK) respectively. Cytospins were then incubated in 4′, 6-diamidino-2-phenylindole (DAPI, Invitrogen, Paisley, UK) to act as a fluorescent nuclear counter stain. Single label immunohistochemistry detection by light microscopy was also performed on sputum cytospins; these were fixed and incubated with GRM20 primary antibody as described above, and GR was detected using biotinylated goat anti-rabbit IgG secondary antibody (Vector Labs, Peterborough, UK) in conjunction with an avidin–biotin peroxidase complex (Vector Labs, Peterborough, UK) and diaminobenzidine (DAB) substrate. Cell nuclei were counterstained with Mayer's haematoxylin (Sigma, Poole, UK). 2.5. Cell culture Peripheral blood and sputum neutrophils were isolated from 7 COPD patients as described above. Cells were cultured in the presence or absence of dexamethasone 0.1, 1, 10, 100 and 1000 nM (SigmaAldrich, Poole, Dorset) for 24 h. Peripheral blood neutrophils were also stimulated with 100 ng/ml LPS (Sigma-Aldrich, Poole, Dorset) for 24 h, and dexamethasone added to stimulated and unstimulated cells. Supernatants were removed and stored immediately at −80 °C. Levels of supernatant TNFα and CXCL8 were measured by

Table 1 Patient demographics from each study. Cell Culture

N Age (Range) Cigarette pack years FEV1% predicted FEV1/FVC Number on ICS

Immunofluorescence

Immunofluorescence

Sputum cytospin

Lung tissue

COPD

COPD

Non-Smokers

COPD

Non-Smokers

7 62.7 (56–71) 39.4 (9.9) 66 (6.6) 57.1 (5.5) 3

7 67 (59–75) 53 (25.6) 61.8 (12.2) 58.5 (7.4) 6

7 65.3 0 105.6 (23.4) 77.8 (4.9) 0

11 67.5 (55–74) 46.95 (27.5) 68.5 (6.1) 53.4 (15.9) 7

7 62.8 (24–76) 0 115.5 (13.9) 77.0 (8.4) 0

Data shown as mean (SD) unless otherwise stated. ICS = Inhaled corticosteroids.

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ELISA (R&D systems, Abingdon, UK). The lower limits of detection were 15.63 pg/ml and 31.25 pg/ml respectively. For a subset of patients (n = 3) cells were removed at 4 and 24 hour post culture to assess cell viability by trypan blue exclusion. Cytospins were also prepared to determine the effect of cell culture on neutrophil apoptosis. Morphological criteria applied were the disappearance of chromatin bridges between nuclear lobes (early apoptosis), and shrinkage or fragmentation of the nucleus (late apoptosis) [14]. 2.6. Image analysis GR expression in alveolar macrophages was examined in at least 200 CD68 + macrophages per patient. GR expression in CD3 + T cells

was calculated within the sub-epithelial layer of 2 small airways (airways devoid of cartilage and glandular tissue, and with an internal perimeter less than 6 mm) per patient. Due to the low subepithelial numbers of neutrophils, total populations of NE + neutrophils (sub-epithelial and parenchymal) were examined for GR expression. GR expression was assessed in at least 100 NE + neutrophils per sputum cytospin. Sputum macrophages (as determined by morphology) were analysed for GR expression following single label immunhistochemistry; at least 100 macrophages per sputum cytospin were counted. To obtain dual label images, fluorescent images from the same field were captured as greyscale images then a dye tint applied according to filter set used. Digital micrographs were obtained using a Nikon Eclipse 80i microscope equipped with

Fig. 1. Glucocorticoid receptor expression by airway neutrophils — Peripheral lung tissue.Representative images of dual-label immunofluorescence for glucocorticoid receptor (GR) (A, D, and G Green) and neutrophil elastase (B, E and H Red) with composite images (C, F and I) from 11 COPD patients. Analysis of sub-epithelial neutrophils near a small bronchiole (A–C). Note the presence of a GR−NE+ neutrophil (arrow). Analysis of parenchymal neutrophils (D–F). Note the presence of a GR+NE+ neutrophil (arrow), identified by the bright orange colour due to the coexpression of red and green. Analysis of parenchymal neutrophils (G–I), note the presence of GR negative neutrophils (arrow) in the presence of GR positive alveolar macrophages (G, *). Representative images showing negative control staining that displays levels of autofluorescence in parenchymal lung tissue (J–L). All sections were counterstained with the nuclear dye DAPI (Blue). Insets in C, F and I are increased magnification of the boxed area. The antibody used in these experiments detects both GR α and GR β isofoms.

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a QImaging digital camera and ImagePro Plus 5.1 software (MediaCybernetics, Marlow, UK). Quantification of individual cell counts, length of airway luminal perimeter and parenchymal area was carried out using the ImagePro plus 5.1 software. All micrographs were stored as tiffs and were cropped, where applicable, using Adobe Photoshop 6.0. 2.7. Statistics Data was analysed in GraphPad Prism and statistics performed in GraphPad Instat (GraphPad Software, San Diego, CA, USA; http:// www.graphpad.com). ELISA and immunohistochemistry data were normally distributed. For lung tissue ANOVA was used to compare GR expression between cell types, and when p b 0.05 subsequent unpaired t tests were performed to compare expression between groups. Unpaired t tests were used to analyse sputum GR expression between groups. Analysis of steroid response within groups was performed using repeated measures ANOVA with Tukey post test. Onetailed paired t-tests were used to compare the effects of dexamethasone on cytokine production between sputum and blood neutrophils.

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71.4%) and CD3 + cells expressing GR (COPD mean 58.1% and NS mean 45.9%) was higher than that observed in neutrophils (Fig. 2). The differences in the percentage of cells expressing GR between cell types were statistically significant in both COPD patients and controls (p b 0.0001 by ANOVA). Representative dual-label immunofluorescence images for GR expression in alveolar macrophages and sub-epithelial CD3 cells are shown in Fig. 3A–C and D–F respectively. GR was detected within the epithelial cells of small airways from all samples, with representative images shown in Fig. 3G–I. GR distribution within lung tissue was similar in 4 COPD patients when the monoclonal anti-GR (Clone-41) which is specific for GR α isoform was substituted for the polyclonal GRM20 antibody (Fig. 3J–L). GR α negative neutrophils were clearly seen in the presence of positive alveolar macrophages (Fig. 3L). No immunoreaction product was observed when the GR primary antibody was omitted from the protocol, or substituted with an isotoype control antibody, on adjacent serial sections. Dual-label immunofluorescence demonstrated that 100% of peripheral blood neutrophils from 3 NS expressed GR (Fig. 4E shows a representative example from 1 subject).

3. Results

3.2. GR expression within sputum neutrophils

3.1. GR expression within peripheral lung tissue

We investigated GR expression using the GRM20 antibody that detects both GR α and GR β isoforms in sputum cytospins from 7 COPD patients and 7 NS. In both subject groups, the majority of GR expression was confined to cells that morphologically resembled macrophages and squamous cells (Fig. 4A and B). Dual-label immunofluorescence demonstrated that the mean percentage of neutrophils that expressed GR was low; 3.8% from COPD patients (Fig. 4C) and 6.9% for NS (Fig. 4D), with no difference between groups (p= 0.35). GR was found to be expressed on 67.4% of sputum macrophages in COPD patients following single label immunohistochemistry.

We studied GR expression in peripheral lung tissue from 11 COPD patients and 7 NS using the GRM20 antibody that detects both GR α and GR β isoforms. Infiltrating neutrophils within the lung parenchyma and sub-epithelial region had low levels of GR expression in both COPD patients and NS. Representative dual-label immunofluorescence images for neutrophils from COPD patients are shown in Fig. 1; A–C shows sub-epithelial neutrophils lacking GR expression, while D–F shows parenchymal neutrophils similarly lacking GR expression, however a single positive neutrophil is highlighted with the arrow. G–I shows GR negative neutrophils in the presence of GR positive alveolar macrophages (identified by cell morphology). A low percentage of neutrophils expressed GR; means 6.8% and 4.3% in COPD patients and NS respectively, with no difference between groups (p = 0.09). The percentage of neutrophils expressing GR was low in all subjects (Fig. 2). We also investigated GR expression within peripheral lung CD3 + T-cells and CD68 + alveolar macrophages. The percentage of alveolar macrophages expressing GR (COPD mean 73.7% and NS mean

Fig. 2. Quantification of glucocorticoid receptor expression by lung macrophages, T cells and neutrophils.Each dot represents the final mean percentage of glucocorticoid receptor (GR) positive cell count for each subject for each cell type, while the horizontal bar represents the overall group mean. Numbers within brackets represent the total number of cells analysed. Reduced neutrophil GR expression within each group was significant compared to the other cell types (p b 0.0001 for both COPD and NS). No difference was observed for cell type specific GR expression between groups (p > 0.05 for neutrophils, macrophages and lymphocytes).

3.3. Cell culture Sputum differential cell counts for the 7 COPD patients who produced samples for cell cultures are presented in Table 2. The enrichment procedure increased the percentage of neutrophils in cell culture to 89%. Previous studies have shown and we observed (data not shown) that LPS had no effect on isolated sputum neutrophils [15]. Unstimulated TNFα and CXCL8 production from sputum neutrophils was 560 pg/ml and 3559 pg/ml respectively. TNFα and CXCL8 from unstimulated peripheral blood neutrophils were 639 pg/ml and 2352 pg/ ml respectively. LPS increased TNFα and CXCL8 release from peripheral blood neutrophils to 2011 pg/ml and 6042 pg/ml respectively. Dexamethasone had only a modest inhibitory effect on TNF and CXCL8 release from sputum cells (Fig. 5), with 41.5% and 39.7% inhibition observed respectively even at high concentrations (1 μM). By comparison, stimulated and unstimulated peripheral blood neutrophils from the same patients were more sensitive to dexamethasone, with inhibitions of 92% and 93.1% (stimulated cells) and 80.4 and 79.9% (unstimulated cells) for TNFα and CXCL8 respectively at 1 μM dexamethasone. The effects of dexamethasone on TNFα were significantly reduced in sputum compared to LPS stimulated blood neutrophils at all concentrations and at 1000 nM in unstimulated peripheral blood neutrophils (Fig. 5A). The inhibition of CXCL8 was significantly lower in sputum neutrophils at all concentrations compared to both stimulated and unstimulated peripheral blood neutrophils (Fig. 5B). The mean cell viability at 24 h, determined by trypan blue exclusion, was 98%, indicating minimal cell necrosis. Neutrophil morphology was also examined after 24 hour cell culture; only a minority of cells displayed signs of late stage apoptosis (19%) (Fig. 4F).

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Fig. 3. Glucocorticoid receptor expression by lung macrophages, T cells and small airway epithelium.Representative photomicrographs of dual-label immunofluorescence for glucocorticoid receptor (GR) (A, D and G Green) and different immune cell phenotypes (B, E, and H Red) with composite images (C, F and I) from 11 COPD patients. All sections were counterstained with the nuclear dye DAPI (Blue). GR was detected within the majority of CD68+ alveolar macrophages (A–C). Detection of GR within sub-epithelial CD3+ T lymphocytes (D–F). GR expression by cytokeratin+ve bronchiole epithelium (G–I). Representative photomicrographs from COPD patients (n=4) of single label immunofluorescence for GRα (Clone 41, Red, J–L) expression within alveolar macrophages (J, arrow), airway epithelium (K, arrow). Light microscopic detection of GRα (Clone 41, Brown) expression within alveolar macrophages in proximity to neutrophils devoid of expression (L, arrows). The antibody that detects both GR α and GR β isofoms was used in A–I, the antibody that detects GR α only was used in J–L. Magnification × 200.

4. Discussion It is known that neutrophil numbers are increased in the lungs of COPD patients [1–4]. We observed that airway neutrophils from both COPD patients and controls had greatly reduced GR expression in contrast to other inflammatory cells. The response of airway neutrophils to dexamethasone was reduced compared to blood neutrophils, where GR expression was high. Our findings suggest that reduced GR expression on airway neutrophils is a mechanism involved in the limited clinical response to inhaled glucocorticoid therapy in COPD patients. GR expression has been described in epithelial cells, smooth muscle, smooth muscle cells, endothelial cells and inflammatory cells in the lungs [11,16,17]. However, we are unaware of any studies that have specifically co-localised GR expression with neutrophil elastase

expression in order to provide quantitative data specifically for neutrophils. We observed high levels of GR expression in peripheral blood neutrophils, suggesting that neutrophils in the lung lose GR expression, in healthy subjects as well as patients with COPD. Our observation of low level GR expression in airway neutrophils in both COPD patients and NS indicates that this is not a disease specific phenomenon, but part of “normal” pathophysiology. However, airway neutrophil numbers are low in healthy subjects, in contrast to increased airway neutrophil numbers in COPD patients [1–4]. Our observation that airway neutrophils from both the lumen and small airways have very low levels of GR expression was consistent in COPD patients and NS. Our sample sizes were modest, but our data is so strikingly consistent in all patients that we believe that any increase in sample size would not alter our findings. The low expression levels of GR were observed in every subject, without any outlying

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Fig. 4. Glucocorticoid receptor expression by airway neutrophils — Sputum.Representative photomicrographs of COPD (n = 7) (A and C) and NS sputum samples (n = 7) (B and D). Single label IHC demonstrates glucocorticoid receptor (GR) expression was mainly expressed by macrophages (A, arrows) and squamous cells (B, *). Dual-label immunofluorescence for GR (Green) and sputum neutrophils (neutrophil elastase, Red) (C and D). Expression of GR (Green) by peripheral blood neutrophils (Red) (E). Following Rapid-Diff staining cell death was identified in only a small number of cells in isolated sputum neutrophils after 24 h in culture (F, arrow). Cells were counterstained with Mayer's haematoxylin (A and B) or the nuclear dye DAPI (Blue) (C–E). The antibody used in these experiments detects both GR α and GR β isofoms.

results with higher expression levels. It is also unlikely that studying smokers with normal lung function would make any difference to our results, as there is no reason to suspect that the GR expression levels on airway neutrophils from these subjects will be any different to the COPD patients or NS. We investigated the anti-inflammatory effects of glucocorticoids using enriched neutrophil cultures from induced sputum and paired peripheral blood neutrophils. The magnitude of effect of dexamethasone on TNFα and CXCL8 production on sputum cells was modest at best, with the maximal effect even at very high dexamethasone concentrations (1 μM) being approximately 40%. Peripheral blood neutrophils displayed a greater response with maximal inhibitions of 80–90%. This difference in response appears to be related to the differing levels of GR available for ligand binding. Sputum cell culture is an established way of investigating anti-inflammatory drug effects [18]. We adapted this method to enrich the neutrophil percentage. The sputum neutrophil percentage was 89%, as some macrophages were present. Unstimulated lung macrophages secrete low levels of

Table 2 Differential cell counts from induced sputum samples used for cell culture.

Total cellsa Total neutrophilsa % Neutrophils Total macrophagesa % Macrophages % Eosinophils % Lymphocytes % Epithelia

Pre-isolation step

Post-isolation step

31.6 (33.4) 23 (23.7) 73.6 (4.6) 7.3 (7.7) 22.5 (4.4) 1.1 (0.9) 0.5 (0.6) 2.4 (2.1)

21.5 19.2 89.6 1.8 8.6 0.5 0.09 1.4

(20.8) (18.3) (2.9) (1.5) (3.4) (0.7) (0.2) (1.5)

Differential cell counts for sputum samples used for cell culture pre- and post- neutrophil isolation step (n=7). Data presented as mean (SD). a Denotes number of cells ×106.

cytokines such as TNFα [19,20]. In contrast, the unstimulated sputum cell cultures that we performed secreted higher levels of cytokines. This suggests that the cytokine secretion in these cultures was predominantly from activated neutrophils. Blood neutrophils have a naturally short life span with a half-life less than 24 h [21]. The rate of constitutive apoptosis, determined by morphology, of peripheral blood neutrophils in vitro has been reported to be 58% at 20 h [22]. We observed that apoptosis of cultured airway neutrophils was markedly lower (19%) at a similar time point, suggestive of a local lung environment that prolongs the natural half-life of these cells. In vitro studies have demonstrated that apoptosis of peripheral blood derived neutrophils can be delayed or inhibited by granulocyte macrophage-colony stimulating factor (GM-CSF) and lipopolysaccharide (LPS) [23–26], both of which may be responsible for prolonging neutrophil survival in the airways. It has also been reported that glucocorticoids can delay peripheral blood neutrophil apoptosis [26]. This may not be relevant to airway neutrophils as these cells lack GR expression so it is unlikely that glucocorticoids can exert an anti-apoptotic effect on these particular cells. Studies have shown reduced [27], similar [28] and increased [29] apoptosis in COPD airway neutrophils compared to controls. We do not believe that altered apoptosis in COPD neutrophils is related to our key finding of reduced GR expression in airway neutrophils, as t we observed similar GR expression both in COPD and healthy airway neutrophils. Furthermore, apoptosis only occurs in a minority of airway neutrophils [27], while we observed that the vast majority of these cells did not express GR. The molecular mechanisms responsible for glucocorticoid resistance in COPD have been the topic of much investigation. Using whole pieces of lung tissue, it has been reported that both GRα and GRβ protein levels are reduced in COPD patients compared to controls [11]. We took a different approach, focusing on cell type specific

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has functional implications in vitro. We hypothesise that our striking observation of low GR expression levels on airway neutrophils and their poor in vitro response to dexamethasone, provides a possible reason why many COPD patients have a poor clinical response to glucocorticoid treatment. Our results highlight the clinical need to find alternative therapeutic approaches for targeting neutrophils in COPD patients. Abbreviations COPD Chronic Obstructive Pulmonary Disease GR glucocorticoid receptor NS Non-smoker TNF Tumour necrosis factor CXCL8 Interleukin–8 NFκB Nuclear factor kappa-light-chain-enhancer of activated B cells GOLD Global Initiative for Chronic Obstructive Lung Disease DTT dithiothreitol DCC differential cell count NE neutrophil elastase PBMC peripheral blood mononuclear cells PMN Polymorphonuclear leukocytes

Competing interests

Fig. 5. Differing responses to dexamethasone by blood (PMN) and sputum neutrophils. Isolated neutrophils (n = 7) were incubated with increasing concentrations of dexamethasone (0.1–1000nM) for 24 h and supernatants analysed for (A) TNFα and (B) CXCL8. Data presented are mean percentage inhibition ± SEM. One-tailed paired t-tests were performed comparing sputum and PMN; a significantly reduced effect of dexamethasone on sputum neutrophils compared to LPS stimulated PMN is denoted by * p b 0.05, ** p b 0.001 and *** p b 0.0001, and compared to unstimulated PMN denoted by # p b 0.05, ## p b 0.001 and ### p b 0.0001.

expression. Previous reports have suggested altered expression of the GRα and GRβ splice variants in neutrophils [30]. The selective loss of signalling GRα in neutrophils, and its replacement with the splice variant GRβ, which lacks ligand binding activity and has dominant negative effects may lead to decreased glucocorticoid efficacy. There are studies documenting increased expression of GRβ in the lungs of patients with corticosteroid resistant [31] and severe asthma [32]. We used an antibody reactive with both α and β forms, as well as an α-specific antibody, and observed reduced expression of the GRα isoforms that was selective to lung neutrophils. This suggests a simple lack of expression rather than an underlying splicing defect in airway neutrophils. Although GR expression is essential to life, little is known about how expression is regulated. In most cell types transcription is driven from a series of promoters that are GC rich, and lie in a CpG island [33]. Attempts to characterise regulatory elements have thus far yielded little useful information, but the nature of the promoter suggests constitutive expression. GR protein is regulated by posttranslational modification, and targeting by the 26S proteosome [34]. Indeed, studies using proteasomal inhibitors enhance GR expression, and transcriptional effects [32]. Therefore, loss of GR expression in neutrophils may be due to transcriptional silencing, as for example by DNA methylation [35], or enhanced protein degradation [36]. 5. Conclusions In this study, we describe for the first time that airway neutrophils have reduced GR expression compared to their peripheral blood counterparts. Moreover we report that this lack of GR expression

JP, KG, BK, BMM, RS, AB and DR have no conflicts of interest to disclose. DS has received sponsorship to attend international meetings, honoraria for lecturing or attending advisory boards, and research grants from various pharmaceutical companies including AstraZeneca, Boehringer Ingelheim, Chiesi, GlaxoSmithKline, Almirall, Forest, Pfizer, UCB, Novartis, and Cipla. Funding This research was funded by an unrestricted research grant from GlaxoSmithKline. The funding source had no involvement in study design; in the collection, analysis, and interpretation of data; in the writing of the paper or in the decision to submit the paper for publication. References [1] O'Donnell R, Peebles C, Ward J, Daraker A, Angco G, Broberg P, et al. Relationship between peripheral airway dysfunction, airway obstruction, and neutrophilic inflammation in COPD. Thorax 2004;59:837–42. [2] Keatings VM, Collins PD, Scott DM, Barnes PJ. Differences in interleukin-8 and tumor necrosis factor-α in induced sputum from patients with chronic obstructive pulmonary disease or asthma. Am J Respir Crit Care Med 1996;153:530–4. [3] Hogg JC, Chu F, Utokaparch S, Woods R, Elliott WM, Buzatu L, et al. The nature of small-airway obstruction in chronic obstructive pulmonary disease. N Eng J Med 2004;350:2645–52. [4] Lapperre TS, Willems LN, Timens W, Rabe KF, Hiemstra PS, Postma DS, et al. Small airways dysfunction and neutrophilic inflammation in bronchial biopsies and BAL in COPD. Chest 2007;131:53–9. [5] Cowburn AS, Condliffe AM, Farahi N, Summers C, Chilvers ER. Advances in neutrophil biology: clinical implications. Chest 2008;134:606–12. [6] Barnes PJ. Emerging pharmacotherapies for COPD. Chest 2008;134:1278–86. [7] Barnes PJ, Ito K, Adcock IM. Corticosteroid resistance in chronic obstructive pulmonary disease: inactivation of histone deacetylase. Lancet 2004;363:731–3. [8] Soriano JB, Sin DD, Zhang X, Camp PG, Anderson JA, Anthonisen NR, et al. A pooled analysis of FEV1 decline in COPD patients randomized to inhaled corticosteroids or placebo. Chest 2007;131:682–9. [9] Pujols L, Mullol J, Torrego A, Picado C. Glucocorticoid receptors in human airways. Allergy 2004;59:1042–52. [10] Marwick JA, Caramori G, Stevenson CS, Casolari P, Jazrawi E, Barnes PJ, et al. Inhibition of PI3Kdelta restores glucocorticoid function in smoking-induced airway inflammation in mice. Am J Respir Crit Care Med 2009;179:542–8. [11] Fujishima S, Takeda H, Kawata S, Yamakawa M. The relationship between the expression of the glucocorticoid receptor in biopsied colonic mucosa and the glucocorticoid responsiveness of ulcerative colitis patients. Clin Immunol 2009;133: 208–17. [12] Globalinitiative for chronic obstructive pulmonary disease spirometry guidelines. http://www.goldcopd.com/OtherResourcesItem.asp?l1=2&l2=2&intId=1836.

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