SUBJECTS

Ten patients with COPD, eight long term smokers with normal lung function, and eight healthy non-smoking volunteers participated in the study. All were men of a similar age (mean (SD) 67 (2) years, 61 (2) years, and 57 (3) years, respectively, p>0.05). Patients with COPD had moderate airflow obstruction (forced expiratory volume in one second (FEV₁) 46.8 (4.2)% predicted value) and mild arterial hypoxaemia (Pao ₂ 9.7 (0.4) kPa) without hypercapnia (Paco ₂ 5.3 (0.2) kPa). All patients were clinically stable, as defined by the absence of any change in their regular treatment and/or need to seek medical attention during the previous 4 months. Patients with bronchial asthma, pneumonia, or lung cancer were excluded. All patients were treated with inhaled bronchodilators but none received inhaled or oral steroids. None of the patients were current smokers. Their smoking history (50 (5) pack years) was similar to that of the smokers with normal lung function (53 (3) pack years). By definition, spirometric measurements were normal in the smokers without COPD (FEV₁ 97.1 (5.4)% predicted value).

All participants gave their written consent, having been fully informed of the nature, risks and potential benefits of the study. The research and ethical review committee of our institution approved the investigation.

STUDY DESIGN

Thirty ml of peripheral venous blood was taken from each participant. Current smokers refrained from smoking for at least 12 hours before blood sampling.8 ,9 From these blood samples neutrophils were harvested (see below) and the respiratory burst and surface expression of adhesion molecules (LFA-1, Mac-1, andl-selectin) in these neutrophils were assessed, both under basal conditions and after stimulation. In addition, in neutrophils obtained from patients with COPD and from long term smokers with normal lung function the mRNA expression of p22-phox and Mac-1 (CD11b) was examined.

LUNG FUNCTION MEASUREMENT

Forced spirometric parameters (GS Warren Collins, USA) were measured according to international guidelines.10Spirometric reference values were those of a Mediterranean population.11 Arterial blood gas tensions (IL BG3, Izasa, Spain) were determined only in patients with COPD.

ISOLATION OF NEUTROPHILS

Neutrophils were isolated from peripheral blood samples according to the methodology previously described in our laboratory.7 Briefly, leucocyte rich plasma was obtained by mixing with an equal volume of endotoxin free Hemoce reagent (Hoechst Iberica, Barcelona, Spain). This was followed by sedimentation during 1 hour at 4°C. Neutrophils were separated from the leucocyte rich plasma by centrifugation on a 15 ml layer of a Ficoll-Paque research grade gradient (Pharmacia Biotech, Uppsala, Sweden) at 900g for 30 minutes at 22°C. Residual erythrocytes were removed by mixing the neutrophil rich pellet with 50 ml of ice cold 0.15 M NH₄Cl solution which was gently agitated at 4°C for 10 minutes and then centrifuged at 750g for 10 minutes at 4°C. The neutrophil pellet was washed once with phosphate buffered saline (PBS) and resuspended with 1 ml PBS, counted by Sysmex K-4500 (Toa Medical Electronics Co Ltd) and adjusted to 4 × 10⁶ cells/ml with PBS. Neutrophils harvested by this technique were >97% pure as assessed by Giemsa staining and 99% viable as assessed by trypan blue exclusion.

MEASUREMENT OF THE NEUTROPHIL RESPIRATORY BURST BY FLOW CYTOMETRY

Activation of the neutrophil respiratory burst was determined by the formation of the fluorescent compound rhodamine-123 from dihydrorhodamine-123 (DHR; Molecular Probes, Eugene, OR, USA).12 In brief, two 200 μl samples of the neutrophil suspension (2 × 10⁶ cells/ml) in polypropylene tubes (Falcon no 2052, Beckton-Dickinson, Lincoln Park, NJ, USA) were incubated with 10 μl of a DHR solution (100 μg/ml) for 10 minutes at 37°C. One sample was used to assess spontaneous ROS production. The other was mixed with 20 μl of a 20 μg/ml phorbol myristate acetate solution (PMA; Sigma Chemical Co, St Louis, MO, USA) and incubated for 15 minutes at 37°C. At the end of this second incubation period 500 μl of cold PBS were added to both samples and these were kept on ice until analysed by flow cytometry on a Beckton-Dickinson FACScan (Beckton-Dickinson, Mountain View, CA, USA) with a gate setting for neutrophils on forward and side scatter. Ten thousand cells were analysed; green fluorescence (FL1) was determined and mean cellular fluorescence intensities (MFI) were calculated usinglysis II software.

ADHESION MOLECULE IMMUNOFLUORESCENCE AND FLOW CYTOMETRIC ANALYSIS

One hundred μl samples of neutrophil suspensions (4 × 10⁶ cells/ml) were mixed with 20 μl of FITC labelled monoclonal antibody anti-LFA-1 (CD11a) (25.3.1 clone, Immunotech, Marseille, France), FITC labelled anti-Mac-1 (CD11b) (BEAR 1 clone, Immunotech), FITC labelled anti-l-selectin (CD62L) (DREG56 clone, Immunotech), and FITC labelled IgG₁ (2T8–2F5 MsIgG₁, Coulter Immunology, Hialeah) that acted as a non-specific control antibody. These were incubated at 4°C for 30 minutes, washed twice in ice cold PBS, resuspended in 1 ml PBS, and kept on ice until analysed. Neutrophils were assessed under resting conditions and after stimulation with 2 ng/ml human tumour necrosis factor α (ΤΝFα; Sigma Chemical Co, St Louis, MO, USA) for 90 minutes at 37°C in 5% CO₂. Cells were assessed with a gate setting for neutrophils on forward and side scatter diagrams and fluorescence on the FL1 channel (530 nm) was collected with the Beckton-Dickinson lysis II software. Ten thousand cell counts were accumulated for the analysis. Non-specific binding (IgG₁) on resting and stimulated neutrophils was measured at MFI values of 2.59 (0.56) and 3.43 (0.94) units, respectively.

RNA ISOLATION AND REVERSE TRANSCRIPTION-POLYMERASE CHAIN REACTION (RT-PCR)

Total RNA was extracted from the neutrophil sample using a single step isolation system (Trizol Reagent, Gibco BRL, Life Technologies, NY, USA) based on the method of Chomczynski and Sacci.13The RNA was precipitated, washed twice with 70% ethanol, and quantified in a spectrophotometer at 260 nm (Shimadzu UV-Visible Recording Spectrophotometer, Shimadzu Corporation, Kyoto, Japan). Two μg of total RNA was reverse transcripted into cDNA using the oligo(dT)₁₈ primer and AMV reverse transcriptase (Promega, Madison, WI, USA). PCR was performed on the resulting cDNA using specific primers for p22-phox (sense primer 5′-GTTTGTGTGCCTGCTGGAGT-3′ and antisense primer 5′-TGGGCGGCTGCTTG ATGGT-3′),14 for CD11b (5′-CAGAGCGT GGTCCAGGCACCA-3′ and 5′-CCTTCAT CCGCCGAAAGTCA-3′),15 and for β-actin (5′-GTGGGGCGCCCAGGCACCA-3′ and 5′-CTCCTTAATGTCACGCACGATTTC-3′)16 using Taq polymerase according to the manufacturer's instructions. The PCR protocol was as follows. For p22-phox: 3 minutes at 94°C, 1 minute at 55°C, 1 minute at 72°C for one cycle; 1 minute at 94°C, 1 minute at 55°C, 1 minute at 72°C for 35 cycles; 1 minute at 94°C, 10 minutes at 60°C for one cycle. For CD11b: 4 minutes at 94°C for one cycle; 30 seconds at 94°C, 30 seconds at 63°C, 1 minute at 72°C for 30 cycles; 5 minutes at 72°C for one cycle. For β-actin: 1 minute at 94°C, 1 minute at 58°C, 2 minutes at 72°C for 35 cycles; 7 minutes at 72°C for one cycle. Following PCR, products were fractionated by 2% agarose gel electrophoresis and stained with ethidium bromide. Gels were subjected to scanner densitometry and the bands were quantified with the aid of the Sigmagel gel analysis software (Jandel Scientific Corporation, San Rafael, CA, USA). The results are expressed as a ratio of β-actin expression, which served as a housekeeping gene.

STATISTICAL ANALYSIS

The results are shown as mean (SE) together with the mean difference between groups and the 95% confidence interval (CI) of this difference. To assess the statistical significance of differences between the three study groups we used analysis of variance (ANOVA) followed by post hoc analysis (Scheffe test) if appropriate. The paired Student's t test was used to investigate the significance of changes within each group after stimulation. A p value of less than 0.05 was considered significant.