Emphysema- and airway-dominant COPD phenotypes defined by standardised quantitative computed tomography.

EvA (Emphysema versus Airway disease) is a multicentre project to study mechanisms and identify biomarkers of emphysema and airway disease in chronic obstructive pulmonary disease (COPD). The objective of this study was to delineate objectively imaging-based emphysema-dominant and airway disease-dominant phenotypes using quantitative computed tomography (QCT) indices, standardised with a novel phantom-based approach.441 subjects with COPD (Global Initiative for Chronic Obstructive Lung Disease (GOLD) stages 1-3) were assessed in terms of clinical and physiological measurements, laboratory testing and standardised QCT indices of emphysema and airway wall geometry.QCT indices were influenced by scanner non-conformity, but standardisation significantly reduced variability (p<0.001) and led to more robust phenotypes. Four imaging-derived phenotypes were identified, reflecting "emphysema-dominant", "airway disease-dominant", "mixed" disease and "mild" disease. The emphysema-dominant group had significantly higher lung volumes, lower gas transfer coefficient, lower oxygen (PO2 ) and carbon dioxide (PCO2 ) tensions, higher haemoglobin and higher blood leukocyte numbers than the airway disease-dominant group.The utility of QCT for phenotyping in the setting of an international multicentre study is improved by standardisation. QCT indices of emphysema and airway disease can delineate within a population of patients with COPD, phenotypic groups that have typical clinical features known to be associated with emphysema-dominant and airway-dominant disease.

The EvA study: aims and strategy.

The EvA study is a European Union-funded project under the Seventh Framework Programme (FP7), which aims at defining new markers for chronic obstructive pulmonary disease (COPD) and its subtypes. The acronym is derived from emphysema versus airway disease, indicating that the project targets these two main phenotypes of the disease. The EvA study is based on the concept that emphysema and airway disease are governed by different pathophysiological processes, are driven by different genes and have differential gene expression in the lung. To define these genes, patients and non-COPD controls are recruited for clinical examination, lung function analysis and computed tomography (CT) of the lung. CT scans are used to define the phenotypes based on lung density and airway wall thickness. This is followed by bronchoscopy in order to obtain samples from the airways and the alveoli. These tissue samples, along with blood samples, are then subjected to genome-wide expression and association analysis and markers linked to the phenotypes are identified. The population of the EvA study is different from other COPD study populations, since patients with current oral glucocorticoids, antibiotics and exacerbations or current smokers are excluded, such that the signals detected in the molecular analysis are due to the distinct inflammatory process of emphysema and airway disease in COPD.

Chemokine expression by small sputum macrophages in COPD.

Small sputum macrophages represent highly active cells that increase in the airways of patients with inflammatory diseases such as chronic obstructive pulmonary disease (COPD). It has been reported often that levels of cytokines, chemokines and pro-teases are increased in sputum supernatants of these patients. In COPD, the small sputum macrophages may contribute to these supernatant proteins and recruit additional cells via specific chemokine expression patterns. We therefore investigated the expression profile of chemokines in sputum macrophages obtained from COPD patients in comparison to cells from healthy donors and cells isolated after inhalation of lipopolysaccharide (LPS). We used the minimally invasive procedure of sputum induction and have purified macrophages with the RosetteSep technology. Using macrophage purification and flow cytometry we show that in COPD small sputum macrophages account for 85.9% ± 8.3% compared with 12.9% ± 7.1% of total macrophages in control donors. When looking at chemokine expression we found, for the small macrophages in COPD, increased transcript and protein levels for CCL2, CCL7, CCL13 and CCL22 with a more than 100-fold increase for CCL13 mRNA (P < 0.001). Looking at active smokers without COPD, there is a substantial increase of small macrophages to 60% ± 15% and, here, chemokine expression is increased as well. In a model of airway inflammation healthy volunteers inhaled 20 µg of lipopolysaccharide (LPS), which resulted in an increase of small sputum macrophages from 18% ± 19% to 64% ± 25%. The pattern of chemokine expression was, however, different with an upregulation for CCL2 and CCL7, while CCL13 was downregulated three-fold in the LPS-induced small macrophages. These data demonstrate that sputum macrophages in COPD show induction of a specific set of CCL chemokines, which is distinct from what can be induced by LPS.

Standardized single-platform assay for human monocyte subpopulations: Lower CD14+CD16++ monocytes in females.

We present a novel single-platform assay for determination of the absolute number of human blood monocyte subpopulations, i.e., the CD14(++)CD16(-) and the CD14(+)CD16(++) monocytes. A four-color combination of antibodies to CD14, CD16, CD45, and HLA-DR reduces the spill-over of natural killer cells and of granulocytes into the CD14(+)CD16(++) monocyte gate. For these CD14(+)CD16(++) monocytes, the intra-assay coefficient of variation (CV) was 4.1% and the inter-assay CV was 8.5%. Looking at a cohort of 40 donors aged 18-60 years, we found no age dependence. There was however an effect of gender in that females had lower CD14(+)CD16(++) monocytes (45.4 +/- 13.5 cells/microl) compared with males (59.1 +/- 20.3 cells/microl) (P < 0.02). Using this novel approach, we can confirm that exercise will lead to more than three-fold increase of the CD14(+)CD16(++) monocytes. Also, we show that therapy with low doses of glucocorticoids will deplete these cells. This robust single-platform assay may be a useful tool for monitoring the absolute number of monocyte subpopulations in health and disease.

Decreased expression of HLA-DQ and HLA-DR on cells of the monocytic lineage in cystic fibrosis.

UNLABELLED: We studied HLA class II molecules on blood monocyte subsets, blood dendritic cells, sputum macrophages, and monocyte-derived macrophages at the protein (flow cytometry) and mRNA level (RT-PCR) in adult patients with cystic fibrosis (CF) and healthy control subjects as putative contributors to the CF phenotype. In healthy donors, we found a high average HLA-DQ expression of 4.35 mean specific fluorescence intensity units (ΔMnI) on classical blood monocytes. In F508del homozygous CF patients, the average ΔMnI was low (1.80). Patients were divided into two groups, in which 14 of these patients had HLA-DQ expression above 2 ΔMnI (average 3.25 ΔMnI, CF-DQ(group1)) and 36 below (average 1.24 ΔMnI, CF-DQ(group2)). Also, the CD16-positive monocyte subset and blood dendritic cells showed much lower levels of HLA-DQ for the CF-DQ(group2) patients compared with healthy controls. In macrophages from sputum and derived from monocytes, in vitro HLA-DQ expression was dramatically decreased to background levels in CF-DQ(group2). MHC class II transcripts were reduced in CF with a sevenfold decrease in HLA-DQß1 for CF-DQ(group2) patients. Higher levels of the inflammation marker CRP were associated with low HLA-DQ protein expression, and in vitro treatment with the inflammatory molecule lipopolysaccharide reduced HLA-DQ expression. Interferon γ (IFNγ) could overcome this effect in healthy donor cells while, in CF, the IFNγ-induced activation was impaired. Our data demonstrate a pronounced reduction of HLA-DQ expression in CF, which is associated with inflammation and a reduced response to IFNγ. KEY MESSAGE: • CF patients show a reduced expression of MHCII molecules in monocytes and macrophages. • HLA-DQ and HLA-DR transcript levels are also reduced in CF patients. • CF patient C-reactive protein levels correlate with low HLA-DQ expression. • Reduced expression of MHC class II molecules appears to be linked to inflammation. • CF patients exhibit an impaired response to IFNgamma.

A defect of CD16-positive monocytes can occur without disease.

The CD16-positive monocytes have been first described in 1988 but to date no selective defect in the number of these cells in blood has been reported. We now describe a family in which three of four siblings lack both CD16-positive monocyte subsets, i.e. the nonclassical and the intermediate monocytes. All three had CD16-positive monocytes of 2 cells/µl or less as compared to 52±18 cells/µl in healthy controls. The index case was affected by recurrent pleural effusion and infections and had evidence of an auto-inflammatory condition but no mutation of any of the relevant candidate genes. The other two siblings without CD16-positive monocytes were apparently healthy. There was no defect in serum M-CSF levels and no mutation in the M-CSF and M-CSFR genes. The data indicate that the absence of CD16-positive monocytes in blood does not lead to disease.

Differential inflammatory response to inhaled lipopolysaccharide targeted either to the airways or the alveoli in man.

Endotoxin (Lipopolysaccharide, LPS) is a potent inducer of inflammation and there is various LPS contamination in the environment, being a trigger of lung diseases and exacerbation. The objective of this study was to assess the time course of inflammation and the sensitivities of the airways and alveoli to targeted LPS inhalation in order to understand the role of LPS challenge in airway disease.In healthy volunteers without any bronchial hyperresponsiveness we targeted sequentially 1, 5 and 20 µg LPS to the airways and 5 µg LPS to the alveoli using controlled aerosol bolus inhalation. Inflammatory parameters were assessed during a 72 h time period. LPS deposited in the airways induced dose dependent systemic responses with increases of blood neutrophils (peaking at 6 h), Interleukin-6 (peaking at 6 h), body temperature (peaking at 12 h), and CRP (peaking at 24 h). 5 µg LPS targeted to the alveoli caused significantly stronger effects compared to 5 µg airway LPS deposition. Local responses were studied by measuring lung function (FEV(1)) and reactive oxygen production, assessed by hydrogen peroxide (H(2)O(2)) in fractionated exhaled breath condensate (EBC). FEV(1) showed a dose dependent decline, with lowest values at 12 h post LPS challenge. There was a significant 2-fold H(2)O(2) induction in airway-EBC at 2 h post LPS inhalation. Alveolar LPS targeting resulted in the induction of very low levels of EBC-H(2)O(2).Targeting LPS to the alveoli leads to stronger systemic responses compared to airway LPS targeting. Targeted LPS inhalation may provide a novel model of airway inflammation for studying the role of LPS contamination of air pollution in lung diseases, exacerbation and anti-inflammatory drugs.

Epigenetic regulation of vitamin D converting enzymes.

While 25-OH-D3 serum levels in humans undergo a large seasonal variation, 1,25-(OH)2-D3 is regulated within a narrow range. We speculate that in addition to the known genomic and nongenomic regulation there could be further epigenetic mechanisms involved. We annotated the human CYP27B1 (alpha-1-hydroxylase) and CYP24A1 (24-hydroxylase) genes for CpG islands and sequenced these in bisulfite treated DNA extracted of peripheral blood lymphocytes from 384 individuals. 40 CpG sites could be analyzed, of these 15 in CYP27B1 and 25 in CYP24A1. The average methylation ratio (MR) in CYP27B1 was 11% (s.d. 5%) with the highest ratio observed in exon 1 (38%). CYP24A1 showed only a 6.5% MR (s.d. 5%). Neither CYP24A1 nor CYP27B1 MR correlated with season of examination date nor with current 25-OH-D3 and 1,25-(OH)2-D3 serum levels except of a weak association of three consecutive CYP27B1 CpG sites and 25-OH-D3 levels. In summary, human PBLs showed only weak methylation in the upstream region of CYP27B1 and none in CYP24A1. As PBLs represent an heterogeneous pool of cells, a further analysis of the seasonal methylation pattern in B or T cell subsets (or other tissues like liver or kidney) is warranted including an extended coverage of the CYP27B1 promotor.

Fractionated exhaled breath condensate collection shows high hydrogen peroxide release in the airways.

BACKGROUND: Exhaled breath condensate (EBC) allows noninvasive monitoring of inflammation in the lung. Activation of inflammatory cells results in an increased production of reactive oxygen species, leading to the formation of hydrogen peroxide (H(2)O(2)). In addition, cigarette smoking causes an influx of inflammatory cells, and higher levels of H(2)O(2) have been found in EBC of smokers. However, there are still unresolved issues reflected by large variations in exhaled H(2)O(2) and uncertainties about the origin of H(2)O(2) release in the lung. METHODS: We collected EBC as fractionated samples from the airways and from the lung periphery in 10 nonsmokers, eight asymptomatic smokers, and in eight chronic obstructive pulmonary disease (COPD) patients, and H(2)O(2) concentration and acidity (pH) were analyzed in the airway and the alveolar fraction. RESULTS: In all subjects studied, H(2)O(2) was 2.6 times higher in the airway versus the alveolar fraction. Airway H(2)O(2) was twofold higher in smokers and fivefold higher in COPD patients compared to nonsmokers. In all study groups, there was no significant difference in deaerated pH between the airway and the alveolar sample. CONCLUSIONS: Exhaled H(2)O(2) is released at higher concentrations from the airways of all subjects studied, implying that the airways may be the dominant location of H(2)O(2) production. Because many lung diseases cause inflammation at different sites of the lung, fractionated sampling of EBC can reduce variability and maintain an anatomical allocation of the exhaled biomarkers.