Maria Tsoumakidou

Dendritic cells in chronic obstructive pulmonary disease - New players in an old game

Dendritic cells (DCs) are professional antigen-presenting cells responsible for immune homeostasis. In the lungs responses to tissue damage or infection, they initiate and orchestrate innate and adaptive immunity. There are immature and mature states and at least three phenotypic and functional subsets. DCs circulate in the blood and localize to mucosal surfaces in immature form where they act as sentinels, sampling constituents of the external environment that breach the epithelium. With internalization of antigen, they are activated, mature, and migrate to draining lymph nodes to induce the proliferation and regulate the balance of Th1/Th2 T cells or to induce a state of tolerance, the last dependent on maturation status, extent of cell surface costimulatory molecule expression, and cytokine release. Cigarette smoke has modulatory effects varying with species, dose, the location examined within the lung, and the marker or technique used to identify DCs. Healthy smokers (and smokers with asthma) have reduced numbers of large airway mature DCs. In chronic obstructive pulmonary disease, the number of immature DCs is increased in small airways, whereas in smokers with chronic obstructive pulmonary disease, the total number of DCs appears to be reduced in large airways. We hypothesize that the long-term effects of cigarette smoke include reduction of DC maturation and function, changes that favor repeated infection, increased exacerbation frequency, and the altered (CD8(+) T-cell predominant) pattern of inflammation associated with this progressive chronic disease.

Isolation of myeloid and plasmacytoid dendritic cells from human bronchoalveolar lavage fluid.

Studies of bronchoalveolar lavage fluid (BALF) dendritic cells (DC) have been hampered by the scarcity of DC and the lack of DC‐specific surface markers. Four surface Ag have been recently described as specific markers for distinct subsets of DC and have been used for the isolation and characterization of fresh noncultured DC from lung resection specimens: BDCA‐1 (CD1c) and BDCA‐3 for myeloid DC type 1 and type 2, respectively, and BDCA‐2 and BDCA‐4 for plasmacytoid DC. The aim of this study was to develop a new method for the isolation of BALF DC, using immunomagnetic separation of BDCA+ cells. Mononuclear cells were obtained from BALF after Ficoll‐Paque density gradient centrifugation. Monocytes, T cells and B cells were magnetically labelled and depleted. The unlabelled cell fraction was incubated with BDCA‐1, BDCA‐3 and BDCA‐4 beads and the total BDCA+ DC were retained. The ability of isolated DC to induce T‐cell responses was evaluated by coculturing the isolated DC with immunomagnetically sorted naive T cells. The above procedure resulted in a population of viable DC that showed a strong capacity in induce T‐cell responses. Functionally intact human BALF myeloid DC types 1 and 2 as well as plasmacytoid DC can be easily obtained by immunomagnetic isolation. Considering that bronchoalveolar lavage is a minimally invasive procedure, these cells are optimal candidates with which to elucidate the properties and capabilities of pulmonary DC.

Induced sputum in the investigation of airway inflammation of COPD

During the last decade, the method of sputum induction (SI) has offered the opportunity to study inflammation in patients with chronic obstructive pulmonary disease (COPD). This paper reviews methodological aspects of SI and summarizes its uses in the research of inflammation in COPD, including sputum cellularity and soluble markers. SI is a relatively safe, reliable, and reproducible technique, used to investigate different aspects of airway inflammation. Although various methods of induction and processing have been proved safe and highly reproducible, a generally accepted method is needed. Sputum analysis has given evidence for increased numbers of macrophages and neutrophils in COPD patients compared to normal subjects. In some studies, increased numbers of eosinophils have been also reported. Changes in various mediators have been found in sputum supernatant of COPD patients (IL-8, LTB-4 and TNF-a). The clinical usefulness of the method in the follow-up of the disease has not been explored extensively. A number of observations in patients with different clinical characteristics could be proven useful in identifying patterns of inflammation associated with different prognosis. Finally, SI could also guide treatment; such as, sputum eosinophilia in COPD could predict response to inhaled corticosteroids.

CXCL13 production in B cells via Toll-like receptor/lymphotoxin receptor signaling is involved in lymphoid neogenesis in chronic obstructive pulmonary disease.

RATIONALE Little is known about what drives the appearance of lymphoid follicles (LFs), which may function as lymphoid organs in chronic obstructive pulmonary disease (COPD). In animal infection models, pulmonary LF formation requires expression of homeostatic chemokines by stromal cells and dendritic cells, partly via lymphotoxin. OBJECTIVES To study the role of homeostatic chemokines in LF formation in COPD and to identify mechanism(s) responsible for their production. METHODS Peripheral lung homeostatic chemokine and lymphotoxin expression were visualized by immunostainings and quantified by ELISA/quantitative reverse transcriptase-polymerase chain reaction in patients with COPD with and without LFs. Expression of lymphotoxin and homeostatic chemokine receptors was investigated by flow cytometry. Primary lung cell cultures, followed by ELISA/quantitative reverse transcriptase-polymerase chain reaction/flow cytometry, were performed to identify mechanisms of chemokine expression. Polycarbonate membrane filters were used to assess primary lung cell migration toward lung homogenates. MEASUREMENTS AND MAIN RESULTS LFs expressed the homeostatic chemokine CXCL13. Total CXCL13 levels correlated with LF density. Lung B cells of patients with COPD were important sources of CXCL13 and lymphotoxin and also expressed their receptors. Cigarette smoke extract, H2O2, and LPS exposure up-regulated B cell-derived CXCL13. The LPS-induced increase in CXCL13 was partly mediated via lymphotoxin. Notably, CXCL13 was required for efficient lung B-cell migration toward COPD lung homogenates and induced lung B cells to up-regulate lymphotoxin, which further promoted CXCL13 production, establishing a positive feedback loop. CONCLUSIONS LF formation in COPD may be driven by lung B cells via a CXCL13-dependent mechanism that involves toll-like receptor and lymphotoxin receptor signaling.

A Prospective Analysis of 184 Hemoptysis Cases – Diagnostic Impact of Chest X-Ray, Computed Tomography, Bronchoscopy

Background:The clinical presentation of hemoptysis often raises a number of diagnostic possibilities. Objectives:This study was designed to evaluate the relative frequency of different causes of hemoptysis and the value of chest radiography, computed tomography (CT) scanning and fiber-optic bronchoscopy in the evaluation of a Greek cohort population. Methods:We prospectively followed a total of 184 consecutive patients (137 males/47 females, 145 smokers/39 nonsmokers) admitted with hemoptysis between January 2001 and December 2003 to the University Hospital of Heraklion. Follow-up data were collected on August 2005. Results:The main causes of hemoptysis were bronchiectasis (26%), chronic bronchitis (23%), acute bronchitis (15%) and lung cancer (13%). Bronchiectasis was significantly more frequent in nonsmokers (p < 0.02). Among nonsmokers, patients with moderate/severe bleeding or a history of tuberculosis were more likely to have bronchiectasis (OR 8.25; 95% CI 1.9–35.9, p = 0.007 and OR 16.5; 95% CI 1.7–159.1, p = 0.007, respectively). Nonsmokers with normal or abnormal X-rays were equally likely to have bronchiectasis (OR 2.5; 95% CI 0.66–9.39, p = 0.2). Lung cancer was only found in smokers. Smokers with normal X-rays were less likely to have lung cancer compared to smokers with abnormal X-ray (OR 5.4; 95% CI 1.54–19.34, p = 0.004). There were no smokers with normal CT and lung cancer. Follow-up data were collected in 91% of patients. Lung cancer did not develop in any patient assumed to have hemoptysis of another origin than lung cancer on initial evaluation. Conclusions:Bronchiectasis is the main diagnosis in patients admitted with hemoptysis to a Greek University Hospital and it is more frequent among nonsmokers with moderate/severe bleeding and/or previous tuberculosis infection. Nonsmokers with moderate/severe hemoptysis and/or a history of tuberculosis should be evaluated with high-resolution CT. Smokers with hemoptysis are at increased risk for lung cancer and need to be extensively evaluated with chest CT and bronchoscopy.

Decreased Small Airway and Alveolar CD83+ Dendritic Cells in COPD

BACKGROUND Dendritic cells (DCs) have been reported to be increased in the small airways of patients with COPD, but the maturity status of these cells is unclear. We have quantified the numbers of cells expressing markers associated with DC maturation. METHODS Lung tissue was obtained at resection for lung cancer from 41 patients with COPD (30 current smokers and 11 ex-smokers; 32 steroid-treated patients and 9 steroid-naïve patients), 19 ex-smokers without COPD and 9 never-smokers without COPD. Tissue sections were immunostained for CD1a to mark immature DCs, and for CD83, fascin, and DC-lysosome-associated membrane protein (DC-LAMP) to delineate mature DCs. RESULTS The volume density (ie, the volume of DCs as the percentage volume of the airway wall) comprising CD83+ DCs was significantly reduced in patients with COPD (median, 0; range, 0 to 5.1%) vs smokers (median, 2.8%; range, 0 to 10.2%) and never-smokers (median, 1.9%; range, 0.8 to 5.1%) without COPD (p = 0.000 and 0.012, respectively). Using a semiquantitative score for the alveolar wall, CD83+ DCs also were decreased in patients with COPD (median, 0; range, 0 to 2%) vs smokers (median, 1%; range, 0 to 2%) and never-smokers (median, 1%; range, 0.7 to 2%) without COPD (p = 0.004 and 0.04, respectively). No differences were detected in CD83+ DCs between current smokers and ex-smokers with COPD or between steroid-treated and steroid-naive patients. No differences were detected in CD1a+ DCs. Fascin and DC-LAMP were found to have poor specificity for mature DCs. CONCLUSIONS COPD is associated with decreased numbers of (mature) CD83+ DCs in small airways and alveoli. The relevance of such a reduction on pulmonary immune responses requires further investigation.

Innate immunity proteins in chronic obstructive pulmonary disease and idiopathic pulmonary fibrosis

ABSTRACT Chronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF) may be caused by epithelial cell injury. Epithelial cells respond to injury by secreting innate immunity proteins. To investigate whether altered levels of innate immunity proteins are observed in COPD and IPF, the authors assessed secretory leukocyte protease inhibitor (SLPI), elafin, CC16, and β-defensin-2 levels by enzyme-linked immunosorbent assay (ELISA) in sputum supernatants from COPD patients (n = 19), smokers without COPD (n = 21), and never-smokers (n = 10) and in BALF supernatants from patients with IPF (n = 11) and subjects without IPF (n = 11). CC16 levels were decreased, whereas SLPI and elafin levels were increased in COPD patients (0.8 [0–4.2] μg/mL, 2.5 [0.3–10.5] μg/mL, 213 [152–318] pg/mL, respectively) compared to smokers without COPD (1.8 [0.1–21.2] μg/mL, 0.8 [0.2–2.6] μg/mL, 172 [71–473] pg/mL, respectively) and never-smokers (0.5 [0–4.8] μg/mL, 0.1 [0.05–0.6] μg/mL, 188 [129–218] pg/mL, respectively) (CC16: P = .001; SLPI: P <.001; elafin: P = .041). β-Defensin-2 was detected in smokers without COPD (98 [10–729] pg/mL) and never-smokers (74 [35–410] pg/mL), but not in COPD. SLPI and elafin levels did not differ between IPF patients and controls, but CC16 levels were increased in IPF (0.5 [0–2.3] versus 0.2 [0–0.3] μg/mL; P = .019). β-Defensin-2 was not detected in BALF. In conclusion, in COPD, secretion of CC16 and β-defensin-2 might be suppressed, whereas SLPI and elafin secretion is up-regulated. In IPF, only CC16 secretion is up-regulated.

Inflammatory cell profiles and T-lymphocyte subsets in chronic obstructive pulmonary disease and severe persistent asthma

Background Severe persistent asthma (SPA) and chronic obstructive pulmonary disease (COPD) are both associated with non‐reversible airflow limitation and airway neutrophilia.

Decreased sputum mature dendritic cells in healthy smokers and patients with chronic obstructive pulmonary disease.

Background: Asthmatics who smoke have decreased pulmonary mature dendritic cells (DCs). Chronic obstructive pulmonary disease (COPD) patients have an increased amount of pulmonary immature DCs. We hypothesized that healthy smokers and patients with COPD have decreased pulmonary mature DCs. Methods: We identified sputum DCs expressing the maturation markers CD83 and DC-lysosome associated membrane protein (DC-LAMP) and DC subpopulations (i.e. myeloid and plasmacytoid DCs) by flow cytometry in healthy smokers before they entered a smoking cessation trial (n = 30), in the same smokers after 6 months of smoking cessation (n = 11) and in COPD patients (n = 28, 14 current and 14 ex-smokers). 12 healthy never-smokers served as controls. DC numbers were expressed as percentage of total sputum CD45+ leukocytes. Results: CD83+ and DC-LAMP+ mature DCs were decreased in healthy smokers before they ceased smoking compared to after (p = 0.003 and p = 0.049, respectively) and in smokers before smoking cessation compared to never-smokers (p = 0.027 and p = 0.028, respectively). COPD patients, both current and ex-smokers, showed decreased CD83+ mature DCs compared to never-smokers and smokers after cessation (p = 0.042 and p = 0.004, respectively). Conclusions: Cigarette smoking and COPD per se are associated with a decrease in pulmonary mature DCs. We speculate that this reduction is involved in the immunopathogenesis of smoking-related respiratory disorders, such as COPD.

Novel insights into the aetiology and pathophysiology of increased airway inflammation during COPD exacerbations.

Airway inflammation increases during acute exacerbations of COPD. Extrinsic factors, such as airway infections, increased air pollution, and intrinsic factors, such as increased oxidative stress and altered immunity may contribute to this increase. The evidence for this and the potential mechanisms by which various aetiological agents increase inflammation during COPD exacerbations is reviewed. The pathophysiologic consequences of increased airway inflammation during COPD exacerbations are also discussed. This review aims to establish a cause and effect relationship between etiological factors of increased airway inflammation and COPD exacerbations based on recently published data. Although it can be speculated that reducing inflammation may prevent and/or treat COPD exacerbations, the existing anti-inflammatory treatments are modestly effective.