ABSTRACT
Chronic obstructive pulmonary disease (COPD) is smoking-related and associated with increased cytotoxic CD8+ T-cells in the airway. There is a wide range of susceptibility to the damaging effects of cigarette smoke with only a small proportion of smokers progressing to COPD. We have previously reported increased intracellular Th1 cytokines in blood, BAL and intraepithelial CD8+T cells in current and ex-smokers with COPD, whereas healthy smokers showed localized Th1 response in the lung only. We thus hypothesised that Th1-associated chemokines or their receptors on CD8+T-cells may be differentially expressed in the blood of healthy smokers, current smoker COPD subjects and those who had ceased smoking. We investigated chemokines, chemokine receptors and Th1 and cytotoxic T-cell markers in blood and BAL using flow cytometry, ELISA and cytometric bead array. In blood, CXCR3, CCR4, intracellular CCR3 and the Th1 marker 62L(-)CD45RO(+) were increased in both COPD groups and healthy smokers. In contrast, cytotoxic T-cells, ITAC, MIG, IFN-gamma and CCR5 were increased in both COPD groups but not smokers. In BAL, the Th1 marker 62L(-)CD45RO(+), CCR5, CXCR3, IFN-gamma, RANTES, IL-8, MCP-1, MIG and ITAC were increased in both COPD groups and smokers versus controls. Our findings are consistent with systemic inflammation in COPD associated with an increased influx of cytotoxic and Th1 cells into the airway. The differential expression of specific chemokines and their receptors in blood from COPD subjects and healthy smokers suggests that inclusion of these markers in any panel designed for the non-invasive investigation of smokers with a disposition to COPD would be clinically relevant.
Subject(s)
Chemokines/metabolism , Pulmonary Disease, Chronic Obstructive/metabolism , Receptors, Chemokine/metabolism , Smoking/metabolism , Th1 Cells/immunology , Adult , Aged , Aged, 80 and over , Antigens, CD/analysis , Bronchoalveolar Lavage Fluid , Flow Cytometry , Humans , Interferon-gamma/metabolism , Lymphocyte Count , Middle Aged , Pulmonary Disease, Chronic Obstructive/immunology , Smoking/immunology , T-Lymphocytes, Cytotoxic/immunology , T-Lymphocytes, Cytotoxic/metabolism , Young AdultABSTRACT
Increased bronchial epithelial cell apoptosis and CD8+ T-cell numbers in the blood and airways have been reported in COPD. These cells can induce apoptosis via the granzyme-b/perforin-mediated pathway. We hypothesized that increased levels of granzyme-b/perforin would be detected in COPD, contributing to apoptosis and tissue damage. Intracellular granzyme-b/perforin were measured in blood-derived T-cells and natural killer (NK) cells from COPD subjects (30 current and 30 ex-smokers), 20 asymptomatic current-smokers and 30 never-smokers, and bronchoalveolar lavage (BAL)-derived T-cells from a cohort of these subjects using flow cytometry. Soluble granzyme-b was determined by ELISA. In blood, there was an increased percentage of T-cells expressing intracellular granzyme-b/perforin for both COPD groups but not asymptomatic smokers (versus never-smokers). Soluble granzyme-b was undetectable. In BAL, soluble granzyme-b levels and the percentage of T-cells expressing intracellular granzyme-b/perforin were increased in both COPD groups and asymptomatic smokers. There was a significant correlation between granzyme-b expression in BAL and apoptosis of bronchial epithelial cells. Most circulating NK cells expressed granzyme-b/perforin, with the median fluorescence intensity of staining increased in both COPD groups and asymptomatic smokers. Granzyme-mediated apoptosis may thus be one mechanism of lung injury in COPD. The changes that persist despite smoking cessation in COPD likely reflect pathophysiological changes in COPD as opposed to the effects of smoking per se.
Subject(s)
Bronchi/pathology , Granzymes/metabolism , Membrane Glycoproteins/metabolism , Pore Forming Cytotoxic Proteins/metabolism , Pulmonary Disease, Chronic Obstructive/metabolism , Smoking , Adult , Aged , Apoptosis , Biomarkers/metabolism , Bronchi/metabolism , Bronchoalveolar Lavage Fluid/chemistry , Bronchoalveolar Lavage Fluid/cytology , CD8-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/metabolism , CD8-Positive T-Lymphocytes/pathology , Disease Progression , Enzyme-Linked Immunosorbent Assay , Epithelial Cells/pathology , Female , Humans , Intracellular Fluid/metabolism , Male , Middle Aged , Perforin , Prognosis , Pulmonary Disease, Chronic Obstructive/pathology , Severity of Illness Index , T-Lymphocytes, Cytotoxic/immunology , T-Lymphocytes, Cytotoxic/metabolism , T-Lymphocytes, Cytotoxic/pathologyABSTRACT
There were over 600 antibodies submitted to HLDA8, with many of unknown specificity. Of these, 101 antibodies were selected for a blind panel study that also included 5 negative controls and 27 positive controls of known CD specificity making a total of 133 antibodies in the final panel. Of the 101 unknowns, 31 antibodies were identified during the course of this blind panel study as being specific for known molecules and included some specific for MHC class II antigens, CD45 isoforms and the Dombrock antigen. Several antibody pairs among those in the blind panel were found to have very similar staining patterns and were therefore compared by immunohistochemical and/or Western blot analyses for identity.
