Leukocyte Function in Asthma and COPD
Leukocyte Function in Asthma and COPD
The aim of this study is to examine the inflammatory mechanisms involved in the pathogenesis
 of inflammatory lung disease, in particular to compare the inflammatory profile seen in
 asthma and COPD. Evidence for inflammation in asthma and COPD is based on the finding of
 increased numbers of macrophages and neutrophils in the lungs and respiratory secretions of
 these patients. The inflammatory cells produce proteases, as well as, reactive oxidant
 species resulting in a protease/anti-protease imbalance which favours lung destruction. The
 aim is to examine the inflammatory mediators released by inflammatory cells (such as,
 macrophages and lymphocytes) in order to determine whether there are differences between
 non-smoking subjects, smoking subjects and patients with asthma or COPD. Monocytes are
 precursors of alveolar macrophages, and both monocytes and neutrophils are recruited to the
 lung from the blood via the action of specific chemoattractants. We have evidence that in
 inflammation there are higher levels of these chemoattractants. Therefore these cells might
 also demonstrate the same changes seen in alveolar macrophages from these patients.

 We also aim to assess the role of the macrophage precursor (monocyte) and neutrophils in the
 blood. We will also assess lymphocyte/monocyte interaction. We will do this as the lymphocyte
 may be involved in the initial recruitment of inflammatory cells. We will also assess the
 role of cytokines involved with monocyte/macrophage/neutrophil migration in induced sputum as
 well as the role of induced sputum in the migration of monocytes and neutrophils into the
 lung. Our aim is to link the initial changes in blood to the changes causing disease in the
 lungs. We aim to examine cellular responses in four groups of subjects, namely (i)
 non-smoking controls, (ii) smokers without clinical evidence of COPD or asthma, (iii) smokers
 with COPD (iv) asthmatic patients.
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.
Inclusion Criteria:

 Asthmatic patients:

 1. Age 21-79 years of both sexes (females will be taking adequate contraceptive
 measures).

 2. Increase in FEV1 >15% and >200ml following beta2 agonist inhalation, either at the
 time of study or previously documented

 COPD patients:

 1. Stable patients with a post-salbutamol FEV1 30-70% predicted normal of >1L

 2. Increase in FEV1 < 15% and < 200 ml following beta2 agonist inhalation, either at the
 time of study or previously documented

 3. Age 21-79 years of both sexes (females will be taking adequate contraceptive measures
 )

 4. Smokers

 5. No history of allergic or respiratory disease.

 Normal Volunteers

 1. Age 21-79 years of both sexes (females will be taking adequate contraceptive measures
 )

 2. Non-smokers

 3. Normal lung function

 4. No upper respiratory tract infection within the last 4 weeks

 5. No history of allergic or respiratory disease.

 Healthy Smokers 1. Age 21-79 years of both sexes (females will be taking adequate
 contraceptive measures ) 2. Smokers 3. Normal lung function 4. No upper respiratory tract
 infection within the last 4 weeks

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 Exclusion Criteria:

 1. Asthmatic patients with FEV1 less than 40% predicted value

 2. Alcohol abuse

 3. Any history or evidence of hepatic, cardiovascular or renal disease

 4. Any history or evidence of neuropsychiatric disease

 5. Drug abuse or any other condition associated with poor compliance

 6. Pregnancy or breast feeding

 7. Patients are unable to provide written informed consent

 COPD patients:

 1. Any other active lung diseases 2. Upper respiratory infection within the last 4 weeks 3.
 Pregnancy or breast feeding 4. Any mental condition rendering the patient unable to
 understand the nature, scope and possible consequences of the study 5. Subjects unable to
 give informed consent

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