Leukocyte Function in Asthma and COPD

Overall subjects will be characterised into one of four groups: normal non-smoking subjects, healthy smokers, patients with asthma and patients with COPD. Subjects will have had to fulfil inclusion and exclusion criteria, and give written consent.

Recruitment Methods. Volunteer subjects (healthy non-smokers and healthy smokers) will be recruited from within the National Heart and Lung Institute. Volunteers will be recruited via advertising using ethically approved advertisements. Asthmatic and COPD patients will be recruited from the outpatient clinic at The Royal Brompton Hospital. At the screening visit, the diagnosis of asthma or COPD will be confirmed from the clinical case notes. If the patient is a volunteer, they will undergo a full history and examination, spirometry. Each subject will attend on one study visit only. At the first visit a number of investigations will be undertaken including, spirometry with reversibility (400 microg ventolin via metered dose inhaler if initial spirometry shows an obstructive picture to exclude asthmatics), and sputum induction. In addition, 50ml of venous blood will be taken.

More specifically, the following interventions will be undertaken by each subject

1. Medical History and Physical Examination A full medical history and physical examination (including height and weight) will be performed on the initial screening visit, in addition to that recorded in the clinical case notes.
2. Respiratory Assessments Forced Expiratory Volume per second (FEVI) will be measured using a dry wedge spirometer (Vitalograph, Buckingham, UK) and expressed as a percent of predicted value. Subjects will be trained in the use of the apparatus before beginning the study. The baseline value at each visit will be measured after at least fifteen minutes of quiet rest, and will be taken as the highest of three readings made at one minute intervals. 400 microg ventolin administered via a metered dose inhaler will be given if initial spirometry shows an obstructive picture (FEV1 <80% predicted, FEV1 /FVC<70%) in order to distinguish between the presence of asthma or COPD.

3. Sputum Induction Sputum induction will be performed in the Asthma Laboratory. The subject will inhale 3.5% saline at room temperature, nebulised via a De Vilbiss 99 Ultrasonic nebuliser set, at maximum output using a mouthpiece. After five minutes the subject will rinse his/her mouth thoroughly with water, and will be asked to cough deeply and expectorate sputum into a pot. Samples from this first five minute collection are discarded. This procedure will continue for a further ten minutes, and the resulting sample will be collected in a polypropylene pot.

   It is important to explain to the subject that the aim is to obtain sputum, and that saliva should be disposed of by spitting into a bowl which is later discarded.

   In the case of any chest discomfort or shortness of breath, the procedure is stopped and spirometry is repeated. If the FEV1 has fallen by 10% of the predicted value, the subject can rest until the FEV1 has returned to the baseline value or the procedure can be terminated.

   Sputum samples will be analysed for differential and absolute cell counts (eosinophils, neutrophils, macrophages, lymphocytes, and columnar epithelial cells). Sputum supernatants will be assayed for TNF-alpha, IL-8, interferon-gamma, and other cytokine levels.

   Immunohistochemistry will be performed on the induced sputum samples to identify the cellular localisation of factors involved with leukocyte function.

   Cytospin preparations will be made for differential cell counts and the soluble fraction will be used for cytokine assays.

4. Venous Blood Sampling Fifty (50) millilitres of blood shall be venesected at the study visit. We will investigate factors involved in leukocyte function and chemotaxis. We will also assess cellular distributions of receptors thought to be important in regulation by using FACS analysis.