Identification of a distinct glucocorticosteroid-insensitive pulmonary macrophage phenotype in patients with chronic obstructive pulmonary disease Kirandeep.

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Presentation transcript:

Identification of a distinct glucocorticosteroid-insensitive pulmonary macrophage phenotype in patients with chronic obstructive pulmonary disease Kirandeep K. Chana, BSc, PhD, Peter S. Fenwick, BSc, MSc, Andrew G. Nicholson, MD, Peter J. Barnes, FRS, FMedSci, Louise E. Donnelly, PhD Journal of Allergy and Clinical Immunology Volume 133, Issue 1, Pages 207-216.e11 (January 2014) DOI: 10.1016/j.jaci.2013.08.044 Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 1 Cell-surface receptor expression of macrophages isolated from the 30% to 40% (vol/vol) Percoll interface from the lung tissue of nonsmokers. Macrophages were isolated at the 30% to 40% (vol/vol) Percoll interface from the lung tissue of nonsmokers, and cells were incubated with APC–HLA-DR, FITC-CD40, PerCP CY5.5–CD163, PE-CD206, APC-CY7–CD14, and Pacific blue–CD16 for 45 minutes at 4°C and analyzed by using flow cytometry. The percentage of cells isolated from the Percoll interfaces of 30% to 40% (vol/vol) are presented as means ± SEMs (n = 3). The figure is representative of 3 experiments. Journal of Allergy and Clinical Immunology 2014 133, 207-216.e11DOI: (10.1016/j.jaci.2013.08.044) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 2 Cell-surface receptor expression of macrophages isolated from the 30% to 40% (vol/vol) Percoll interface from the lung tissue of smokers. Macrophages were isolated at the 30% to 40% (vol/vol) Percoll interface from the lung tissue of nonsmokers, and cells were incubated with APC–HLA-DR, FITC-CD40, PerCP CY5.5–CD163, PE-CD206, APC-CY7–CD14, and Pacific blue–CD16 for 45 minutes at 4°C and analyzed by using flow cytometry. The percentage of cells isolated from the Percoll interfaces of 30% to 40% (vol/vol) are presented as means ± SEMs (n = 3). The figure is representative of 3 experiments. Journal of Allergy and Clinical Immunology 2014 133, 207-216.e11DOI: (10.1016/j.jaci.2013.08.044) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 3 Cell-surface receptor expression of macrophages isolated from the 30% to 40% (vol/vol) Percoll interface from the lung tissue of patients with COPD. Macrophages were isolated at the 30% to 40% (vol/vol) Percoll interface from the lung tissue of nonsmokers, and cells were incubated with APC–HLA-DR, FITC-CD40, PerCP CY5.5–CD163, PE-CD206, APC-CY7-CD14, and Pacific blue–CD16 for 45 minutes at 4°C and analyzed by using flow cytometry. The percentage of cells isolated from the Percoll interfaces of 30% to 40% (vol/vol) are presented as means ± SEMs (n = 3). The figure is representative of 3 experiments. Journal of Allergy and Clinical Immunology 2014 133, 207-216.e11DOI: (10.1016/j.jaci.2013.08.044) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 4 A-J, Expression of cell-surface markers on macrophages isolated from different Percoll interfaces. Macrophages were isolated from resected human lung tissue from nonsmokers (NS; n = 3), smokers (S; n = 3), and patients with COPD (n = 3) at the interfaces of 30% to 40% (vol/vol; open bars), 40% to 50% (vol/vol; gray bars), and 50% to 60% (vol/vol; black bars) Percoll. Cells were blocked with IgG for 15 minutes at room temperature and incubated with the relevant antibody for 45 minutes at 4°C. Cells were washed and resuspended in FACS fix. Data are presented as means ± SEMs. *P < .05, **P < .01, and ***P < .001 for differences from nonsmokers. #P < .05, ##P < .01, and ###P < .001 for differences from smokers. Journal of Allergy and Clinical Immunology 2014 133, 207-216.e11DOI: (10.1016/j.jaci.2013.08.044) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 5 Release of active MMP-9 from lung tissue macrophages. Macrophages were isolated from the 30% to 40% (vol/vol; A), 40% to 50% (vol/vol; B), and 50% to 60% (vol/vol; C) interfaces of the Percoll gradient and seeded into cell-culture plates. MMP-9 release from all cell fractions was measured by using a fluorometric activity assay: nonsmokers (NS; n = 5, open bars), smokers (S; n = 5, gray bars), and patients with COPD (n = 3-5, black bars). Data are presented as means ± SEMs. *P < .05. Journal of Allergy and Clinical Immunology 2014 133, 207-216.e11DOI: (10.1016/j.jaci.2013.08.044) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 6 Effect of budesonide on LPS-stimulated cytokine release from lung tissue macrophages. Macrophages were isolated from the 30% to 40% (vol/vol; A, D, and G), 40% to 50% (vol/vol; B, E, and H), and 50% to 60% (vol/vol; C, F, and I) interfaces of the Percoll gradient. Cells were pretreated with budesonide at the concentrations indicated for 1 hour, followed by LPS stimulation (10 ng/mL) for 20 hours, and medium was harvested. TNF-α panels (Fig 6, A-C), CXCL8 (Fig 6, D-F), and IL-10 (Fig 6, G-I) were measured by using ELISA, and data were then normalized to LPS stimulation (100%). Triangles, Nonsmokers (n = 6); solid circles, smokers (n = 11); open circles, patients with COPD (n = 7). Data are presented as means ± SEMs. Journal of Allergy and Clinical Immunology 2014 133, 207-216.e11DOI: (10.1016/j.jaci.2013.08.044) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E1 Gating strategy for multiparameter flow cytometry. Dead cells, doublets, and CD3+CD1a+ cells were excluded, and the remaining HLA-DR+ cells were subsequently analyzed for CD14 and CD16, followed by CD163, CD40, and CD206. Journal of Allergy and Clinical Immunology 2014 133, 207-216.e11DOI: (10.1016/j.jaci.2013.08.044) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E2 Reastain Quick-Diff staining of tissue macrophages. Nuclei stained dark blue, and cytoplasm stained light blue. Cell fractions were extracted from the following Percoll interfaces: A, 30% to 40% (vol/vol); B, 40% to 50% (vol/vol); and C, 50% to 60% (vol/vol). Photomicrographs are representative of 6 tissue samples. Journal of Allergy and Clinical Immunology 2014 133, 207-216.e11DOI: (10.1016/j.jaci.2013.08.044) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E3 Expression of CD68 by tissue macrophages. Cytospin preparations were prepared with human lung macrophages. Cells were fixed and stained by using an immunocytochemistry kit with either a CD68 antibody or an isotype control. Slides were then visualized with a light microscope. Cellular staining (haemalum) is purple/blue, and CD68 staining is red/brown. Results are representative of 4 separate experiments. Journal of Allergy and Clinical Immunology 2014 133, 207-216.e11DOI: (10.1016/j.jaci.2013.08.044) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E4 Cell-surface receptor expression of macrophages isolated from the 40% to 50% (vol/vol) Percoll interface from the lung tissue of nonsmokers. Macrophages were isolated at the 40% to 50% (vol/vol) Percoll interface from the lung tissue of nonsmokers, and cells were blocked with IgG for 15 minutes at room temperature and then incubated with APC–HLA-DR, FITC-CD40, PerCP CY5.5–CD163, PE-CD206, APC–CY7-CD14, and Pacific Blue–CD16 for 45 minutes at 4°C. Cells were washed, resuspended in FACS fix, and analyzed by means of flow cytometry. HLA-DR+ cells were selected, followed by CD14+CD16−, CD14−CD16−, and CD14+CD16+ cells. Each of these CD14 and CD16 populations were then analyzed for expression of CD163, CD40, and CD206. The percentage of cells isolated from the Percoll interfaces of 40% to 50% (vol/vol) are presented as means ± SEMs (n = 3). The figure is representative of 3 experiments. Journal of Allergy and Clinical Immunology 2014 133, 207-216.e11DOI: (10.1016/j.jaci.2013.08.044) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E5 Cell-surface receptor expression of macrophages isolated from the 50% to 60% (vol/vol) Percoll interface from the lung tissue of nonsmokers. Macrophages were isolated at the 50% to 60% (vol/vol) Percoll interface from the lung tissue of nonsmokers, and cells were blocked with IgG for 15 minutes at room temperature and then incubated with APC−HLA-DR, FITC-CD40, PerCP CY5.5−CD163, PE-CD206, APC−CY7-CD14, and Pacific Blue−CD16 for 45 minutes at 4°C. Cells were washed, resuspended in FACS fix, and analyzed by using flow cytometry. HLA-DR+ cells were selected, followed by CD14+CD16−, CD14−CD16−, and CD14+CD16+ cells. Each of these CD14 and CD16 populations were then analyzed for expression of CD163, CD40, and CD206. The percentage of cells isolated from the Percoll interfaces of 50% to 60% (vol/vol) are presented as means ± SEMs (n = 3). The figure is representative of 3 experiments. Journal of Allergy and Clinical Immunology 2014 133, 207-216.e11DOI: (10.1016/j.jaci.2013.08.044) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E6 Cell-surface receptor expression of macrophages isolated from the 40% to 50% (vol/vol) Percoll interface from the lung tissue of smokers. Macrophages were isolated at the 40% to 50% (vol/vol) Percoll interface from the lung tissue of smokers, and cells were blocked with IgG for 15 minutes at room temperature and then incubated with APC–HLA-DR, FITC-CD40, PerCP CY5.5–CD163, PE-CD206, APC–CY7-CD14, and Pacific blue–CD16 for 45 minutes at 4°C. Cells were washed, resuspended in FACS fix, and analyzed by using flow cytometry. HLA-DR+ cells were selected, followed by CD14+CD16−, CD14−CD16−, and CD14+CD16+ cells. Each of these CD14 and CD16 populations were then analyzed for expression of CD163, CD40, and CD206. The percentage of cells isolated from the Percoll interfaces of 40% to 50% (vol/vol) are presented as means ± SEMs (n = 3). The figure is representative of 3 experiments. Journal of Allergy and Clinical Immunology 2014 133, 207-216.e11DOI: (10.1016/j.jaci.2013.08.044) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E7 Cell-surface receptor expression of macrophages isolated from the 50% to 60% (vol/vol) Percoll interface from the lung tissue of smokers. Macrophages were isolated at the 50% to 60% (vol/vol) Percoll interface from the lung tissue of smokers, and cells were blocked with IgG for 15 minutes at room temperature and then incubated with APC–HLA-DR, FITC-CD40, PerCP CY5.5–CD163, PE-CD206, APC–CY7-CD14, and Pacific blue–CD16 for 45 minutes at 4°C. Cells were washed, resuspended in FACS fix, and analyzed by using flow cytometry. HLA-DR+ cells were selected, followed by CD14+CD16−, CD14−CD16−, and CD14+CD16+ cells. Each of these CD14 and CD16 populations were then analyzed for expression of CD163, CD40, and CD206. The percentage of cells isolated from the Percoll interfaces of 50% to 60% (vol/vol) are presented as means ± SEMs (n = 3). The figure is representative of 3 experiments. Journal of Allergy and Clinical Immunology 2014 133, 207-216.e11DOI: (10.1016/j.jaci.2013.08.044) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E8 Cell-surface receptor expression of macrophages isolated from the 40% to 50% (vol/vol) Percoll interface from the lung tissue of patients with COPD. Macrophages were isolated at the 40% to 50% (vol/vol) Percoll interface from the lung tissue of patients with COPD, and cells were blocked with IgG for 15 minutes at room temperature and then incubated with APC–HLA-DR, FITC-CD40, PerCP CY5.5–CD163, PE-CD206, APC–CY7-CD14, and Pacific blue–CD16 for 45 minutes at 4°C. Cells were washed, resuspended in FACS fix, and analyzed by using flow cytometry. HLA-DR+ cells were selected, followed by CD14+CD16−, CD14−CD16−, and CD14+CD16+ cells. Each of these CD14 and CD16 populations were then analyzed for expression of CD163, CD40, and CD206. The percentage of cells isolated from the Percoll interfaces of 40% to 50% (vol/vol) are presented as means ± SEMs (n = 3). The figure is representative of 3 experiments. Journal of Allergy and Clinical Immunology 2014 133, 207-216.e11DOI: (10.1016/j.jaci.2013.08.044) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E9 Cell-surface receptor expression of macrophages isolated from the 50% to 60% (vol/vol) Percoll interface from the lung tissue of patients with COPD. Macrophages were isolated at the 50% to 60% (vol/vol) Percoll interface from the lung tissue of smokers, and cells were blocked with IgG for 15 minutes at room temperature and then incubated with APC–HLA-DR, FITC–CD40, PerCP CY5.5–CD163, PE-CD206, APC–CY7-CD14, and Pacific blue–CD16 for 45 minutes at 4°C. Cells were washed, resuspended in FACS fix, and analyzed by using flow cytometry. HLA-DR+ cells were selected, followed by CD14+CD16−, CD14−CD16−, and CD14+CD16+ cells. Each of these CD14 and CD16 populations were then analyzed for expression of CD163, CD40, and CD206. The percentage of cells isolated from the Percoll interfaces of 50% to 60% (vol/vol) are presented as means ± SEMs (n = 3). The figure is representative of 3 experiments. Journal of Allergy and Clinical Immunology 2014 133, 207-216.e11DOI: (10.1016/j.jaci.2013.08.044) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions