T Lymphocyte Insensitivity to Corticosteroids in COPD
T Lymphocyte Insensitivity to Corticosteroids in COPD
14 COPD patients, 10 smokers with normal lung function and 10 healthy non smokers (HNS) were recruited (demographics shown in Table 1). Some of these patients were recruited specifically to undergo a bronchoscopy for research purposes (6 COPD patients, 3 smokers and 7 HNS). The remaining patients were undergoing clinical investigational bronchoscopies for the following reasons; haemoptysis (3 COPD patients, 1 smoker, 3 HNS), unexplained shortness of breath or weight loss (2 COPD patients, 2 smokers), and investigation of abnormal chest X-ray findings suggestive of lung cancer (3 COPD patients and 4 smokers). Subjects with any other pulmonary conditions including history of asthma were excluded. For immunofluorescent analysis, 10 COPD patients and 11 smokers with normal lung function, undergoing surgical resection for suspected or confirmed lung cancer were recruited. Tissue blocks were preselected for this study based on the presence of at least one inflammatory follicle. COPD patients were diagnosed based on a history of smoking (> 10 pack years), typical symptoms and airflow obstruction (FEV1 < 80% predicted, and FEV1/FVC ratio < 0.7). All subjects gave written informed consent. The study was approved by the local research ethics committee.
BAL was collected from the upper lobes, or a lobe not affected by radiographic or endobronchial abnormalities: The bronchoscope was wedged in the bronchus and a maximum of 4 × 60 ml aliquots of pre-warmed sterile 0.9% NaCl solution were instilled into each lobe. The aspirated fluid was stored on ice before filtration (100 μm filter, Becton Dickenson). The filtrate was centrifuged (400 g/10 min at 4°C) and the cell pellet resuspended in RPMI 1640 medium supplemented with 2 mM L-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin. Viable cell counts were determined by trypan blue exclusion (Neubauer hemocytometer) and adjusted to 1 × 10 cells/ml. Macrophages were depleted by adherence by 90 minute incubation in a 24 well plate at 37°C, 5% CO2. The non adherent cell count was adjusted to 2 × 10 cells/ml in supplemented RPMI 1640 medium with additional 10% (vol/vol) fetal calf serum. This cell suspension was then used for cell culture.
1 × 10 cells (non adherent BAL), were seeded in a 96 well plate +/- dexamethasone (1, 0.1 & 0.01 μM) for 2 hours at 37°C, 5% CO2 in 200 μl RPMI-1640 media supplemented with 2 mM L-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin and 10% FCS (v/v). Subsequently lymphocytes were activated by addition of PHA and phorbol myristate acetate (PMA), 10 μg/ml each. Cells were further incubated for 24 hours. This time point was chosen based on initial time course experiments over 2 days in 3 smoking subjects (data not shown). Supernatants (180 μl) were harvested by plate centrifugation 10 mins, 400 g, 4°C and transferred to a fresh 96 well plate for storage at -20°C prior to ELISA analysis.
Release of interleukin (IL)-2 and interferon (IFN) γ was assayed on cell culture supernatants (either undiluted or up to 1:5 dilution with RPMI containing 10% FCS as required) using R&D systems ELISA duosets according to manufacturer's instructions.
Tissue blocks were obtained from an area of the lung as far distal to the tumour as possible, and processed as described previously. Lung tissues were cut into 4-μm sections and lifted onto a polysine coated glass slide. For heat induced epitope retrieval in 0.01 M trisodium buffer pH 6 the lung sections were microwaved for 20 min at 800 W. A cocktail of CD8 with either GRα or GRβ primary antibodies diluted in 1.5% normal serum (Vector Labs, Peterborough, UK) with 0.5% triton × 100 was applied overnight at 4°C. Two CD8 primary antibodies were used to avoid applying a cocktail with two antibodies requiring the same species secondary antibody.
According to primary antibody species requirements, CD8 and GR were detected using either Alexa 568 conjugated goat anti-rabbit immunoglobulin IgG (1:200, Invitrogen, Paisley, UK) and Alexa 488 conjugated goat anti-mouse immunoglobulin IgG secondary antibodies (1:200, Invitrogen) respectively. Sections were counterstained with 4,6-diamidino-2-phenylindole (DAPI, Invitrogen). Omission of primary antibody from staining protocol was used as a negative control.
The number of CD8GRα and CD8GRβ cells within inflammatory follicles were calculated and presented as a percentage of the CD8 population. Digital micrographs were obtained using a Nikon Eclipse 80i microscope (Nikon UK Ltd, Surrey, UK) equipped with a QImaging digital camera (Media Cybernetics, Marlow UK) and ImagePro Plus 5.1 software (Media Cybernetics).
IL-2 and IFNγ data were not normally distributed, and so comparisons between groups were performed using a non-parametric ANOVA. If the ANOVA was significant (p < 0.05), then subsequent Mann Whitney U tests were performed for pairwise comparisons. Data for the percentage inhibition by dexamethasone were parametric. Paired t-tests were used to compare the effects of dexamethasone to PHA/PMA alone. Between group comparisons were performed by ANOVA. If the ANOVA was significant (p < 0.05), then subsequent two way comparisons were performed using an unpaired t-test. Immunofluorescence data were normally distributed; comparisons between groups were performed using unpaired t-tests. All analysis was carried out using GraphPad Prism version 5 (GraphPad Software, Inc., San Diego, CA, USA).
Methods
Study Subjects
14 COPD patients, 10 smokers with normal lung function and 10 healthy non smokers (HNS) were recruited (demographics shown in Table 1). Some of these patients were recruited specifically to undergo a bronchoscopy for research purposes (6 COPD patients, 3 smokers and 7 HNS). The remaining patients were undergoing clinical investigational bronchoscopies for the following reasons; haemoptysis (3 COPD patients, 1 smoker, 3 HNS), unexplained shortness of breath or weight loss (2 COPD patients, 2 smokers), and investigation of abnormal chest X-ray findings suggestive of lung cancer (3 COPD patients and 4 smokers). Subjects with any other pulmonary conditions including history of asthma were excluded. For immunofluorescent analysis, 10 COPD patients and 11 smokers with normal lung function, undergoing surgical resection for suspected or confirmed lung cancer were recruited. Tissue blocks were preselected for this study based on the presence of at least one inflammatory follicle. COPD patients were diagnosed based on a history of smoking (> 10 pack years), typical symptoms and airflow obstruction (FEV1 < 80% predicted, and FEV1/FVC ratio < 0.7). All subjects gave written informed consent. The study was approved by the local research ethics committee.
Cell Collection
BAL was collected from the upper lobes, or a lobe not affected by radiographic or endobronchial abnormalities: The bronchoscope was wedged in the bronchus and a maximum of 4 × 60 ml aliquots of pre-warmed sterile 0.9% NaCl solution were instilled into each lobe. The aspirated fluid was stored on ice before filtration (100 μm filter, Becton Dickenson). The filtrate was centrifuged (400 g/10 min at 4°C) and the cell pellet resuspended in RPMI 1640 medium supplemented with 2 mM L-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin. Viable cell counts were determined by trypan blue exclusion (Neubauer hemocytometer) and adjusted to 1 × 10 cells/ml. Macrophages were depleted by adherence by 90 minute incubation in a 24 well plate at 37°C, 5% CO2. The non adherent cell count was adjusted to 2 × 10 cells/ml in supplemented RPMI 1640 medium with additional 10% (vol/vol) fetal calf serum. This cell suspension was then used for cell culture.
Cell Culture
1 × 10 cells (non adherent BAL), were seeded in a 96 well plate +/- dexamethasone (1, 0.1 & 0.01 μM) for 2 hours at 37°C, 5% CO2 in 200 μl RPMI-1640 media supplemented with 2 mM L-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin and 10% FCS (v/v). Subsequently lymphocytes were activated by addition of PHA and phorbol myristate acetate (PMA), 10 μg/ml each. Cells were further incubated for 24 hours. This time point was chosen based on initial time course experiments over 2 days in 3 smoking subjects (data not shown). Supernatants (180 μl) were harvested by plate centrifugation 10 mins, 400 g, 4°C and transferred to a fresh 96 well plate for storage at -20°C prior to ELISA analysis.
ELISA Assay
Release of interleukin (IL)-2 and interferon (IFN) γ was assayed on cell culture supernatants (either undiluted or up to 1:5 dilution with RPMI containing 10% FCS as required) using R&D systems ELISA duosets according to manufacturer's instructions.
Dual-label Immunofluorescence
Tissue blocks were obtained from an area of the lung as far distal to the tumour as possible, and processed as described previously. Lung tissues were cut into 4-μm sections and lifted onto a polysine coated glass slide. For heat induced epitope retrieval in 0.01 M trisodium buffer pH 6 the lung sections were microwaved for 20 min at 800 W. A cocktail of CD8 with either GRα or GRβ primary antibodies diluted in 1.5% normal serum (Vector Labs, Peterborough, UK) with 0.5% triton × 100 was applied overnight at 4°C. Two CD8 primary antibodies were used to avoid applying a cocktail with two antibodies requiring the same species secondary antibody.
According to primary antibody species requirements, CD8 and GR were detected using either Alexa 568 conjugated goat anti-rabbit immunoglobulin IgG (1:200, Invitrogen, Paisley, UK) and Alexa 488 conjugated goat anti-mouse immunoglobulin IgG secondary antibodies (1:200, Invitrogen) respectively. Sections were counterstained with 4,6-diamidino-2-phenylindole (DAPI, Invitrogen). Omission of primary antibody from staining protocol was used as a negative control.
Image Analysis
The number of CD8GRα and CD8GRβ cells within inflammatory follicles were calculated and presented as a percentage of the CD8 population. Digital micrographs were obtained using a Nikon Eclipse 80i microscope (Nikon UK Ltd, Surrey, UK) equipped with a QImaging digital camera (Media Cybernetics, Marlow UK) and ImagePro Plus 5.1 software (Media Cybernetics).
Data Analysis & Statistics
IL-2 and IFNγ data were not normally distributed, and so comparisons between groups were performed using a non-parametric ANOVA. If the ANOVA was significant (p < 0.05), then subsequent Mann Whitney U tests were performed for pairwise comparisons. Data for the percentage inhibition by dexamethasone were parametric. Paired t-tests were used to compare the effects of dexamethasone to PHA/PMA alone. Between group comparisons were performed by ANOVA. If the ANOVA was significant (p < 0.05), then subsequent two way comparisons were performed using an unpaired t-test. Immunofluorescence data were normally distributed; comparisons between groups were performed using unpaired t-tests. All analysis was carried out using GraphPad Prism version 5 (GraphPad Software, Inc., San Diego, CA, USA).