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• W1970068341 abstract "Chronic obstructive pulmonary disease (COPD) is characterised by chronic inflammation in airways and lung parenchyma. CD8+ T-lymphocytes, crucial effector and regulatory cells in inflammation, are increased in the central and peripheral airways in COPD. The aim of this study was to assess the role of apoptosis in the accumulation of CD8+ T-lymphocytes within the airway wall in COPD. We examined the submucosa of transverse sections of central and peripheral airways from post-operative tissues from non-smokers (n = 16), smokers with normal lung function (n = 16), smokers with mild/moderate COPD (n = 16), and smokers with severe/very severe COPD (n = 9). TUNEL and immunohistochemistry techniques were used to identify apoptosis and cell phenotype, respectively. The percentage of apoptotic CD8+ T-lymphocytes was significantly lower (p < 0.0001) in smokers with mild/moderate COPD than in non-smokers, smokers with normal lung function, and smokers with severe/very severe COPD, and was positively related to values of FEV1 and FEV1/FVC ratio, both in central and in peripheral airways. These data suggest that reduced apoptosis of CD8+ T-lymphocytes may be an important mechanism that contributes to the accumulation of these cells in the airway submucosa in smokers with mild/moderate COPD. Chronic obstructive pulmonary disease (COPD) is characterised by chronic inflammation in airways and lung parenchyma. CD8+ T-lymphocytes, crucial effector and regulatory cells in inflammation, are increased in the central and peripheral airways in COPD. The aim of this study was to assess the role of apoptosis in the accumulation of CD8+ T-lymphocytes within the airway wall in COPD. We examined the submucosa of transverse sections of central and peripheral airways from post-operative tissues from non-smokers (n = 16), smokers with normal lung function (n = 16), smokers with mild/moderate COPD (n = 16), and smokers with severe/very severe COPD (n = 9). TUNEL and immunohistochemistry techniques were used to identify apoptosis and cell phenotype, respectively. The percentage of apoptotic CD8+ T-lymphocytes was significantly lower (p < 0.0001) in smokers with mild/moderate COPD than in non-smokers, smokers with normal lung function, and smokers with severe/very severe COPD, and was positively related to values of FEV1 and FEV1/FVC ratio, both in central and in peripheral airways. These data suggest that reduced apoptosis of CD8+ T-lymphocytes may be an important mechanism that contributes to the accumulation of these cells in the airway submucosa in smokers with mild/moderate COPD. Chronic obstructive pulmonary disease (COPD) is a complex disease characterized by a chronic innate and adaptive inflammatory immune responses to long-term exposure to inhaled toxic gases and particles. Although environmental pollution contributes to this response, cigarette smoke is the major risk factor for the development of COPD, cigarette smokers constituting more than 90% of all COPD patients in developed countries.1Barnes P.J. Chronic obstructive pulmonary disease.N Engl J Med. 2000; 343: 269-280Crossref PubMed Scopus (1211) Google Scholar This inflammatory immune process affects both airways and lung parenchyma, and the cell types mainly involved in this phenomenon include neutrophils, macrophages, and T-cells.2Saetta M. Turato G. Maestrelli P. Mapp C.E. Fabbri L.M. Cellular and structural bases of chronic obstructive pulmonary disease.Am J Respir Crit Care Med. 2001; 163: 1304-1309Crossref PubMed Scopus (450) Google Scholar CD8+ T-lymphocytes appear to play a crucial role in orchestrating the inflammatory response and have been related to disease progression.3Hogg J.C. Chu F. Utokaparch S. Woods R. Elliott W.M. Buzatu L. et al.The nature of small-airway obstruction in chronic obstructive pulmonary disease.N Engl J Med. 2004; 350: 2645-2653Crossref PubMed Scopus (2894) Google Scholar The number of CD8+ T-lymphocytes is increased both in central and in peripheral airways4O’Shaughnessy T.C. Ansari T.W. Barnes N.C. Jeffery P.K. Inflammation in bronchial biopsies of subjects with chronic bronchitis: inverse relationship of CD8+ T lymphocytes with FEV1.Am J Respir Crit Care Med. 1997; 155: 852-857Crossref PubMed Scopus (608) Google Scholar, 5Saetta M. Di Stefano A. Turato G. Facchini F.M. Corbino L. Mapp C.E. et al.CD8+ T-lymphocytes in peripheral airways of smokers with chronic obstructive pulmonary disease.Am J Respir Crit Care Med. 1998; 157: 822-826Crossref PubMed Scopus (666) Google Scholar as well as in lung parenchyma and pulmonary arteries6Saetta M. Baraldo S. Corbino L. Turato G. Braccioni F. Rea F. et al.CD8+ve cells in the lungs of smokers with chronic obstructive pulmonary disease.Am J Respir Crit Care Med. 1999; 160: 711-717Crossref PubMed Scopus (406) Google Scholar in COPD. CD8+ T-lymphocytes represent an essential part of the adaptive immune response to viral infections,7Whitmire J.K. Ahmed R. Costimulation in antiviral immunity: differential requirements for CD4(+) and CD8(+) T cell responses.Curr Opin Immunol. 2000; 12: 448-455Crossref PubMed Scopus (130) Google Scholar therefore the homing of CD8+ T-lymphocytes into the airways might also be promoted by the presence of respiratory viral infections, very common event in patients with COPD,8Ely K.H. Cauley L.S. Roberts A.D. Brennan J.W. Cookenham T. Woodland D.L. Non specific recruitment of memory CD8+ T cells to the lung airways during respiratory virus infections.J Immunol. 2003; 170: 1423-1429PubMed Google Scholar and it might depend on initial activation as well as adhesion and selective chemotaxis9Di Stefano A. Capelli A. Lusuardi M. Caramori G. Balbo P. Ioli F. et al.Decreased T lymphocyte infiltration in bronchial biopsies of subjects with severe chronic obstructive pulmonary disease.Clin Exp Allergy. 2001; 31: 893-902Crossref PubMed Scopus (72) Google Scholar, 10Saetta M. Mariani M. Panina-Bordignon P. Turato G. Buonsanti C. Baraldo S. et al.Increased expression of the chemokine receptor CXCR3 and its ligand CXCL10 in peripheral airways of smokers with chronic obstructive pulmonary disease.Am J Respir Crit Care Med. 2002; 165: 1404-1409Crossref PubMed Scopus (322) Google Scholar of these cells. Despite these observations, the molecular mechanisms by which the CD8+ T-lymphocytes accumulate in the airways of patients with COPD are presently not well understood. Inflammatory cells may accumulate in the airways through a number of mechanisms including increased recruitment from the bloodstream through chemotaxis10Saetta M. Mariani M. Panina-Bordignon P. Turato G. Buonsanti C. Baraldo S. et al.Increased expression of the chemokine receptor CXCR3 and its ligand CXCL10 in peripheral airways of smokers with chronic obstructive pulmonary disease.Am J Respir Crit Care Med. 2002; 165: 1404-1409Crossref PubMed Scopus (322) Google Scholar, 11Chung K.F. Cytokines in chronic obstructive pulmonary disease.Eur Respir J Suppl. 2001; 34: 50s-59sCrossref PubMed Google Scholar and local proliferation12Goya S. Matsuoka H. Mori M. Morishita H. Kida H. Kobashi Y. et al.Sustained interleukin-6 signalling leads to the development of lymphoid organ-like structures in the lung.J Pathol. 2003; 200: 82-87Crossref PubMed Scopus (58) Google Scholar on the one hand, and/or by decreased removal through inefficient clearance by macrophages13Vandivier R.W. Henson P.M. Douglas I.S. Burying the dead: the impact of failed apoptotic cell removal (efferocytosis) on chronic inflammatory lung disease.Chest. 2006; 129: 1673-1682Crossref PubMed Scopus (355) Google Scholar or through reduced apoptosis. In multicellular organisms, apoptosis is essential for development, immune functions, and for the maintenance of homeostasis. Apoptosis is also involved in the control of the “tissue load” of immune effector cells at inflamed sites and it tends to limit inflammatory tissue injury by promoting resolution rather than progression of inflammation.14Haslett C. Savill J.S. Whyte M.K. Stern M. Dransfield I. Meagher L.C. Granulocyte apoptosis and the control of inflammation.Philos Trans R Soc Lond B Biol Sci. 1994; 345: 327-333Crossref PubMed Scopus (208) Google Scholar It has also been shown that decreased or suppressed apoptosis of immune effector cells in inflamed tissues may contribute to chronic inflammation. In rheumatoid arthritis the active suppression of T-cell death by the synovial microenvironment is an important mechanism for the persistence of T-cell infiltrates in chronically inflamed joints.15Salmon M. Scheel-Toellner D. Huissoon A.P. Pilling D. Shamsadeen N. Hyde H. et al.Inhibition of T cell apoptosis in the rheumatoid synovium.J Clin Invest. 1997; 99: 439-446Crossref PubMed Scopus (233) Google Scholar Moreover, delayed eosinophil apoptosis in nasal polyps has been indicated as a novel mechanism by which eosinophils specifically accumulate in polipoid tissue.16Simon H.U. Yousefi S. Schranz C. Schapowal A. Bachert C. Blaser K. Direct demonstration of delayed eosinophil apoptosis as a mechanism causing tissue eosinophilia.J Immunol. 1997; 158: 3902-3908PubMed Google Scholar Our group has previously observed reduced apoptosis of memory T-cells in the airway wall of patients with asthma.17Lamb J.P. James A. Carroll N. Siena L. Elliot J. Vignola A.M. Reduced apoptosis of memory T-cells in the inner airway wall of mild and severe asthma.Eur Respir J. 2005; 26: 265-270Crossref PubMed Scopus (26) Google Scholar However, little is known about survival and apoptosis of inflammatory cells in the airways of patients with COPD. In the present study, we therefore examined the role of apoptosis in the persistent accumulation of CD8+ T-lymphocytes in the airways of patients with COPD. We evaluated this phenomenon by using surgical lung samples, at different levels of the airways and at different stages of the disease. Subjects recruited into the present study were categorized into the following four groups: asymptomatic non-smoking subjects (never smoked) with normal lung function (control non-smokers; n = 16); asymptomatic smokers with normal lung function (control smokers; n = 16); smokers with mild or moderate COPD (n = 16; 5 mild and 11 moderate) and smokers with severe or very severe COPD (n = 9; 3 severe and 6 very severe), based on Global Initiative for Chronic Obstructive Lung Disease (GOLD) criteria18Rabe K.F. Hurd S. Anzueto A. Barnes P.J. Buist S.A. Calverley P. et al.Global Initiative for Chronic Obstructive Lung Disease. Global strategy for the diagnosis, management, and prevention of chronic obstructive pulmonary disease: GOLD executive summary.Am J Respir Crit Care Med. 2007; 176: 532-555Crossref PubMed Scopus (4420) Google Scholar stages I–II and stages III–IV, respectively. Control smokers and smokers with COPD included both current and former smokers. All former smokers had stopped smoking for more than one year. Lung specimens were obtained at surgery either from patients undergoing lobectomy or pneumonectomy for peripheral carcinoma in the mild/moderate COPD cases or control subjects, or from subjects undergoing lung transplantation for emphysema in the severe/very severe COPD cases. During the week before surgery, each subject underwent chest radiography, electrocardiogram, routine blood tests, skin prick tests with common allergen extracts, pulmonary function tests, and an interview regarding respiratory symptoms, medications used and smoking history. All mild/moderate COPD cases used bronchodilators and all severe/very severe COPD cases used, in addition to bronchodilators, regular inhaled corticosteroid therapy and systemic corticosteroid therapy during the frequent exacerbations. During the month preceding surgery, COPD cases had no exacerbations and all subjects had no acute upper respiratory tract infections. All subjects were non-atopic (negative skin prick tests for common allergen extracts) and had no history of asthma or allergic rhinitis. The present study was performed with approval of Sir Charles Gairdner Hospital’s Ethics Committee (Nedlands, Australia), and informed written consent was obtained for each subject prior to surgery. Pulmonary function tests were performed according to standard methods19Standardization of Spirometry, 1994 UpdateAmerican Thoracic Society.Am J Respir Crit Care Med. 1995; 152: 1107-1136Crossref PubMed Scopus (6340) Google Scholar and included measurements of forced expiratory volume in 1 s (FEV1), and forced vital capacity (FVC). Fixed airway obstruction was defined according to recent guidelines20Celli B.R. MacNee W. ATS/ERS Task Force. Standards for the diagnosis and treatment of patients with COPD: a summary of the ATS/ERS position paper.Eur Respir J. 2004; 23: 932-946Crossref PubMed Scopus (3522) Google Scholar as a ratio of FEV1 to FVC of less than 70% predicted, with FEV1 reversibility of less than 12% after inhalation of 200 μg salbutamol. Tissue blocks were taken from transverse central airways and subpleural parenchyma (for peripheral airways) of the lobe obtained at surgery, avoiding areas close to the tumor in control groups and mild/moderate COPD cases who underwent lung resection for carcinoma. The same laboratory procedure was followed for samples from lungs recovered during lung transplantation for severe/very severe COPD cases. Samples were freshly fixed in formalin, dehydrated, and embedded in paraffin wax. 4 μm-thick whole transverse sections were cut, mounted on charged slides, dewaxed, rehydrated, and processed for the detection of apoptosis in CD8+ T-lymphocytes. Apoptosis of CD8+ T-lymphocytes was detected by combining the terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) and the immunohistochemistry techniques to identify cell apoptosis and cell phenotype, respectively.21Bonkhoff H. Fixemer T. Hunsicker I. Remberger K. Simultaneous detection of DNA fragmentation (apoptosis), cell proliferation (MIB-1), and phenotype markers in routinely processed tissue sections.Virchows Arch. 1999; 434: 71-73Crossref PubMed Scopus (20) Google Scholar Sections were first stained by the TUNEL technique which detects DNA strand breaks in cells undergoing apoptosis, using a commercial kit (Roche Applied Science, Penzberg, Germany) following the manufacturer’s instructions. Briefly, terminal deoxynucleotidyl transferase (TdT) was used to incorporate fluorescein-deoxyuridine (dUTP) at sites of DNA cleavage. Incorporated fluorescein was detected by anti-fluorescein antibody conjugated with horse-radish peroxidase (POD). The staining was achieved with 3,3′-diaminobenzidine (DAB)-substrate solution leaving a dark-brown nuclear end-product. Some slides were incubated with DNAse I (Roche Applied Science) (1000 U/ml, 15 min, room temperature) to induce DNA strand breaks or in buffer without TdT enzyme or dUTP as positive and negative controls, respectively. To identify CD8+ T-lymphocytes, the same sections were subsequently stained with a monoclonal mouse anti-human CD8 antibody (MAb) (clone C8/144B; M7103, Dako, Glostrup, Denmark), diluted 1:50. This antibody specifically labels cytotoxic/suppressor T-cells.22Mason D.Y. Cordell J.L. Gaulard P. Tse A.G. Brown M.H. Immunohistological detection of human cytotoxic/suppressor T cells using antibodies to a CD8 peptide sequence.J Clin Pathol. 1992; 45: 1084-1088Crossref PubMed Scopus (61) Google Scholar MAb binding was revealed by labelled streptavidin biotin-alkaline phosphatase method (LSAB2 System-AP; Dako), and New Fuchsin as chromogenic substrate, leaving a red cytoplasmic end-product. To expose the immunoreactive epitope of cell marker, the sections were pre-incubated in Trizma-buffered saline (Sigma–Aldrich, St. Louis, MO, USA), pH 9.1, at 92 °C for 45 min. Negative controls were performed by omitting the primary antibody or by using an isotype control antibody (IgG1) from the same species. Human tonsil tissue was used as a positive control. Sections were finally counterstained with haematoxylin. Airway images were obtained by a light microscope (Leitz Biomed; Leica, Cambridge, UK) at 400× magnification, connected to a video recorder linked to a computerized image system (Quantimet 500 Image Processing and Analysis System, Qwin V0200B software; Leica). In all airway transverse sections, the internal perimeter, defined by the basement membrane perimeter (Pbm), and the submucosa area (region between basement membrane and inner edge of smooth muscle layer) were measured. Airways were assigned to two groups according to their Pbm: central (Pbm>6 mm) and peripheral (Pbm<6 mm).10Saetta M. Mariani M. Panina-Bordignon P. Turato G. Buonsanti C. Baraldo S. et al.Increased expression of the chemokine receptor CXCR3 and its ligand CXCL10 in peripheral airways of smokers with chronic obstructive pulmonary disease.Am J Respir Crit Care Med. 2002; 165: 1404-1409Crossref PubMed Scopus (322) Google Scholar Internal perimeter rather than airway diameter was selected as a criterion to describe airway size because it remains constant irrespective of airway constriction or relaxation.23James A.L. Hogg J.C. Dunn L.A. Paré P.D. The use of the internal perimeter to compare airway size and to calculate smooth muscle shortening.Am Rev Respir Dis. 1988; 138: 136-139Crossref PubMed Scopus (175) Google Scholar Two to five central airways with a full perimeter and two parenchymal sections with approximately six peripheral airways per section were examined for each patient. Airways with a short/long diameter ratio less than one-third were excluded from the study, as being tangentially cut. For each airway section, both apoptotic and non apoptotic CD8+ T-lymphocytes were counted in the submucosa, along the entire length of the basement membrane per unit area (mm2), and apoptotic CD8+ T-lymphocytes were expressed as percentage of apoptotic CD8+ T-lymphocytes over total CD8+ T-lymphocytes in the examined airway submucosa. The final result per subject was the average of the number of apoptotic CD8+ T-lymphocytes present in each airway of that patient. Slides were coded and blindly evaluated by two independent investigators (L.S. and E.P.) without knowledge of clinical and functional data. Double immunofluorescence staining was performed on formalin-fixed paraffin-embedded tissue sections from samples of smoking controls and smokers with mild/moderate COPD to evaluate the co-localization of CD8 and Annexin V or CD4 and Annexin V. Immunostainings were performed using monoclonal mouse anti-human CD8 (clone C8/144B; Dako) or monoclonal mouse anti-human CD4 (clone 4B12; Dako) antibodies. Double labelling was performed using both a secondary fluorescein isothiocyanate (FITC)-conjugated anti-mouse polyclonal rabbit IgG antibody (Sigma–Aldrich) and phycoerythrin (PE)-conjugated Annexin V (BD Biosciences Pharmingen, San Diego,CA, USA) in the same section. Slides were analysed by a fluorescence microscope (Axioscop 2; Zeiss, Heidelberg, Germany). Group data were expressed as mean and SEM or as median and 25th –75th percentiles where appropriate. Analysis of variance (ANOVA) and unpaired Student’s t-test for clinical data, and Kruskall–Wallis test for histological data were used to analyse differences between subject groups. Mann–Whitney U test was performed for comparisons between two groups, if initial Kruskall–Wallis test indicated significance. Spearman’s rank correlation test was used for the correlations between histological and functional data. P value of less than 0.05 was considered as statistically significant. The demographic and clinical characteristics of the four subject groups are shown in Table 1. Sex ratio and mean age were similar in all four groups. As expected from the selection criteria, mild/moderate COPD cases (GOLD stages I/II) had significantly lower values of FEV1 (% predicted) and of FEV1/FVC ratio (%) than smoking controls and non-smoking controls. Moreover, severe/very severe COPD cases (GOLD stages III/IV) had significantly lower values of FEV1 (% predicted) and FEV1/FVC ratio (%) than mild/moderate COPD cases, smoking controls, and non-smoking controls.Table 1Subject characteristics.Control subjects - normal lung functionSmokers with COPDNon-smokersSmokersMild/moderate(GOLD stages I/II)Severe/very severe(GOLD stages III/IV)Subjects examined, n1616169Sex, male/female, n10/611/511/56/3Age, yrs51 ± 648 ± 564 ± 345 ± 3Smoking history, pack/yrscIt was not possible to obtain the precise pack-year history of smoking controls and severe/very severe COPD cases.049 ± 7Current/former smokers, n0/015/111/50/9FEV1, % pred107 ± 490 ± 274 ± 3bSignificantly different from smoking controls and non-smoking controls (p < 0.05).25 ± 4aSignificantly different from mild/moderate COPD cases, smoking controls and non-smoking controls (p < 0.05).FEV1/FVC, %94 ± 284 ± 262 ± 1bSignificantly different from smoking controls and non-smoking controls (p < 0.05).31 ± 2aSignificantly different from mild/moderate COPD cases, smoking controls and non-smoking controls (p < 0.05).Data are expressed as absolute number or mean ± SEM.a Significantly different from mild/moderate COPD cases, smoking controls and non-smoking controls (p < 0.05).b Significantly different from smoking controls and non-smoking controls (p < 0.05).c It was not possible to obtain the precise pack-year history of smoking controls and severe/very severe COPD cases. Open table in a new tab Data are expressed as absolute number or mean ± SEM. The morphometric characteristics of the airways examined are shown in Table 2. In each airway size category there were no statistically significant differences in the mean Pbm among the four subject groups.Table 2Morphometric characteristics of the airways.Control subjects - normal lung functionSmokers with COPDNon-smokersSmokersMild/moderate (GOLD stages I/II)Severe/very severe(GOLD stages III/IV)Pbm, mmaNo statistically significant differences between groups.Central airways15.17 ± 1.2815.26 ± 2.1012.28 ± 1.5212.93 ± 1.05Peripheral airways2.62 ± 0.192.42 ± 0.162.73 ± 0.272.86 ± 0.32Data are expressed as mean ± SEM.Pbm: basement membrane perimeter.a No statistically significant differences between groups. Open table in a new tab Data are expressed as mean ± SEM. Pbm: basement membrane perimeter. Apoptotic CD8+ T-lymphocytes were identified by co-localization of the dark-brown nuclear staining generated by the TUNEL technique with red cytoplasmic staining generated by the CD8 immunoreactivity. Apoptosis was also evaluated on the basis of the morphologic appearance of apoptosis. Specifically, at light microscopy observation, chromatin condensation and nuclear fragmentation were used to identify apoptotic cells (Figure 1, Figure 2).Figure 2High power view of CD8+ T-lymphocytes with CD8 immunoreactivity (red cytoplasmic staining) with A) TUNEL negative nucleus (haematoxylin staining) and B) TUNEL + nucleus (dark-brown nuclear staining). CD8 negative cells with C) TUNEL negative nucleus and D) TUNEL + nucleus. Typical apoptotic morphology is shown in B) and D): shrinkage, chromatin condensation and nuclear fragmentation. Original magnification: X 1000. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)View Large Image Figure ViewerDownload Hi-res image Download (PPT) The median densities of all CD8+ T-lymphocytes, including those undergoing apoptosis, and the median densities of apoptotic CD8+ T-lymphocytes in the submucosa of central and peripheral airways, in the four subject groups, are shown in Table 3. The median percentage of apoptotic CD8+ T-lymphocytes in the submucosa of central airways was significantly lower (p < 0.0001) in mild/moderate COPD cases than in non-smoking controls, smoking controls and severe/very severe COPD cases (median, 25th–75th percentiles: 12, 10–18; 40, 37–46; 36, 33–41; 36, 30–47 respectively) (Fig. 3). No significant differences were found in the percentage of apoptotic CD8+ T-lymphocytes between severe/very severe COPD cases, smoking controls, and non-smoking controls (Fig. 3). Similarly, the median percentage of apoptotic CD8+ T-lymphocytes in the submucosa of peripheral airways was significantly lower in mild/moderate COPD cases than in non-smoking controls or smoking controls (p < 0.0001) and severe/very severe COPD cases (p = 0.0001) (median, 25th–75th percentiles: 17, 12–23; 41, 32–49; 34, 31–39; 34, 30–43 respectively) (Fig. 4).Table 3CD8+ T-lymphocytes in submucosa of the airways.Control subjects - normal lung functionSmokers with COPDNon-smokersSmokersMild/moderate(GOLD stages I/II)Severe/very severe(GOLD stages III/IV)Central airwaysCD8+ T-lymphocytes Total cells/mm219 (11–29)172 (130–227)aSignificantly different from non-smokers (p < 0.0001).182 (99–224)aSignificantly different from non-smokers (p < 0.0001).45 (41–62)bSignificantly different from non-smokers (p = 0.005). Apoptotic cells/mm28 (5–11)61 (51–76)20 (15–31)cSignificantly different from smokers (p < 0.0001).19 (16–23)Peripheral airwaysCD8+ T-lymphocytes Total cells/mm221 (9–40)96 (68–125)aSignificantly different from non-smokers (p < 0.0001).154 (120–245)aSignificantly different from non-smokers (p < 0.0001).82 (42–98)dSignificantly different from non-smokers (p = 0.003). Apoptotic cells/mm27 (3–18)34 (20–51)25 (18–39)26 (20–35)Data are expressed as median and 25th–75th percentiles.a Significantly different from non-smokers (p < 0.0001).b Significantly different from non-smokers (p = 0.005).c Significantly different from smokers (p < 0.0001).d Significantly different from non-smokers (p = 0.003). Open table in a new tab Figure 4Apoptotic (TUNEL+) CD8+ T-lymphocytes in submucosa of peripheral airways in non-smoking and smoking controls, mild/moderate COPD cases and severe/very severe COPD cases. Results are expressed as percent of total CD8+ T-lymphocytes that are apoptotic. Horizontal bars inside boxes represent the median values and limits of boxes represent the 25th and 75th percentiles. P values (inside the figure) represent the results of Mann–Whitney U test analyses. Overall comparison was made by Kruskal–Wallis test (p < 0.0001).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Data are expressed as median and 25th–75th percentiles. Fig. 5, A–C shows a double immunofluorescence analysis from a control smoker demonstrating the presence of Annexin V in CD8+ T-lymphocytes. The number of CD8+ T-lymphocytes that co-localized Annexin V was lower in smokers with mild/moderate COPD than in smoking controls (data not shown). Similar results were observed for the presence of Annexin V in CD4+ T-lymphocytes (Fig. 5, D–F), confirming that the survival of both CD8+ and CD4+ T-lymphocytes is similarly regulated. When all studied subjects were considered together, no significant correlation was observed between apoptosis of CD8+ T-lymphocytes and functional parameters. When severe/very severe COPD cases were excluded from the analysis, significant positive correlations were observed between the percentage of apoptotic CD8+ T-lymphocytes and the values of FEV1 (% predicted) and FEV1/FVC ratio, both in central and in peripheral airways (Fig. 6). Moreover, a significant negative correlation was observed between the total number of CD8+ T–lymphocytes and the percentage of apoptotic CD8+ T-lymphocytes, both in central and peripheral airways (rho = − 0.50, p < 0.0001 and rho = − 0.40, p = 0.002 respectively). This study shows that, in the submucosa of both central and peripheral airways, there is a reduction of the percentage of CD8+ T-lymphocytes undergoing apoptosis in smokers with mild to moderate COPD compared with non-smokers and smokers with normal lung function, and cases of severe/very severe COPD, and this result correlates with the degree of airflow obstruction. To our knowledge, this is the first study examining apoptosis of inflammatory cells in the entire inner wall in transverse sections of both central and peripheral airways in COPD. In the present study, CD8+ T-lymphocyte apoptosis was evaluated by the TUNEL technique and morphological analysis. Different complex pathways are involved in cell apoptosis and therefore different regulatory factors (pro- or anti-apoptotic proteins) might be quantified. The expression of such markers might correlate with the occurrence of apoptosis but it does not precisely measure the ongoing apoptosis. We used here the TUNEL technique because it is the most useful technique to detect cell apoptosis in paraffin-embedded tissues and it has been used and validated in many studies.17Lamb J.P. James A. Carroll N. Siena L. Elliot J. Vignola A.M. Reduced apoptosis of memory T-cells in the inner airway wall of mild and severe asthma.Eur Respir J. 2005; 26: 265-270Crossref PubMed Scopus (26) Google Scholar, 24Bruno A. Chanez P. Chiappara G. Siena L. Giammanco S. Gjomarkaj M. et al.Does leptin play a cytokine-like role within the airways of COPD patients?.Eur Respir J. 2005; 26: 398-405Crossref PubMed Scopus (86) Google Scholar In addition, this technique offers the major advantages of revealing early DNA breaks during apoptosis, measuring the number of apoptotic cells and simultaneously identifying, by immunohistochemistry, the phenotype of the cells undergoing apoptosis within a heterogeneous population.25Sgonc R. Wick G. Methods for the detection of apoptosis.Int Arch Allergy Immunol. 1994; 105: 327-332Crossref PubMed Scopus (134) Google Scholar We" @default.
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• W1970068341 title "Reduced apoptosis of CD8+ T-Lymphocytes in the airways of smokers with mild/moderate COPD" @default.
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