Protease Regulation in COPD: An Immunohistochemical Analysis of COPD Tissue for Glucocorticosteroid Receptor Expression
Chronic Obstructive Pulmonary Disease is strongly associated with cigarette smoking. COPD is described as a poorly reversible decline in lung function. The commonest pathological significance of the disease is thickening of the airway walls that result from proliferation and activation of fibroblast, a process known as fibrosis. The activated fibroblasts releases pro-inflammatory cytokines and induce additional inflammatory cell responses that lead to development of cell pathology. The treatment of COPD with steroid is currently widely ineffective. However, there is theoretical suggestion that the insensitivity to steroid treatment is due to activity at cellular level though the concept needs to be investigated fully through empirical-based studies. In this study, the immunohistochemical (IHC) techniques were used to characterize the expression of glucocorticosteroid receptor(s) using antibodies. The lung tissue samples (resections) obtained from patients with the respiratory disease was embedded in paraffin. The analysis of the findings involved the determination of correlation between morphological appearance of the lung resections and patient demographics and also included lung function and smoking pack year history.
Chronic Obstructive Pulmonary Disease (COPD) is a collective term for progressive lung diseases that include chronic bronchitis, emphysema, and chronic obstructive airways disease (Vestbo 2013). The general symptoms of all these diseases include breathing difficulties due to narrowing of airways (airflow obstruction), increased breathlessness during activity, persistent coughing accompanied with phlegm, and frequent infection of the chest (Singh et al. 2013). It is widely accepted that smoking is the cause and risk factor of developing COPD. The rate of progression once one acquires the disease depends on the extent of exposure (Blackler 2007). For instance, the likelihood of developing COPD is increased with increased smoking and the duration of the behavior. Smoking is both the risk and etiological factor because it induces irritation and inflammation of the lungs, which results to scarring that is known to induce the development of COPD. Note that smoking for a long period increases consistency of inflammation leading to permanent changes in the lung (Blackler 2007). These changes cause the airways to thicken and produce more mucus. These changes incur damage to the delicate walls of the alveoli leading to the development of emphysema and loss of normal elasticity of the lungs (Singh et al. 2008). As the airways become narrowed and scarred the patient exhibit symptoms of breathlessness, coughing, and phlegm (Wright & Churg 2008). Apart from smoking other etiological and risk factors of COPD development and progression include fumes, dust, air pollution, and genetic disorders though they are practically rare (Nannini et al. 2012; Cheyne et al. 2013).
COPD has been identified as one of the commonest respiratory diseases in the United Kingdom. The significant age for disease onset in the UK has been identified to be 35 years (Simpson et al. 2010). However, most of the people with the condition are diagnosed at about 50 years of age (Vos et al. 2012). The prevalence of COPD in the UK is about 3 million people of which only about 900, 000 have been diagnosed (Rycroft et al. 2012). Most people with the condition have not sought any medication such as diagnosis because they often ignore the symptoms such as coughing (Decramer et al. 2013). Analysis based on gender has identified that more men than women are at higher risk of developing COPD. It should be noted that seeking early diagnosis is important step to successful management and treatment of the disease (Cave & Hurst 2011). In this regard, people at risk exhibiting any of the commonest symptoms should seek diagnosis and subsequent management and treatment. Note that COPD is usually diagnosed after consultation with the doctor and other simple test such as breathing tests. Seeking early treatment following once suspicious symptoms start to show enable the patient to receive appropriate treatment and advice that can help to either stop or slow the progression of the disease (Wright & Churg 2008). Before recommendation for diagnosis, the doctor has to establish some information about the exhibited symptoms such as the duration the patient has had the symptoms, whether the patient smokes or has been exposed to other risk factor, and history of smoking among others (Wang et al. 2012). Tests carried out during consultation may include the use of stethoscope to examine the breathing condition of the chest and the determination of body mass index (BMI) by measuring the weight and height. Additional examination before recommendation for diagnosis may include the determination of how well the lungs functions using the spirometery (Shafazand 2013). Spirometry test is one of the most important steps to diagnosis since it determines the breathing test by recoding two measurements. First is the volume of air that an individual can breathe out in 1 second. This measurement is referred to as forced expiratory volume in 1 second (FEV1) (Wright & Churg 2008). Second is the total amount of air breathed out. The measurement is referred to as forced vital capacity (FVC). Consistent reading is important and this can be measured using the device to measure the two breathing parameters a few times (Wright & Churg 2008). The effectiveness of the reading can be enhanced by comparing with normal measurements for the patient’s age, which helps to identify if the airway has been obstructed (Mammen & Sethi 2012). The confirmation by the above tests helps the doctor to recommend other test such as chest X-ray, blood test, electrocardiogram (ECG) and echocardiogram, peak flow test, blood oxygen level, computerized tomography (CT) scan, and blood test for alpha-1-antitrypsin deficiency (Wright & Churg 2008). COPD is a type of obstructive lung disease that is characterized by almost irreversible poor air flow and air trapping. Poor air flow has been identified to causing breakdown of lung tissue, a condition known as emphysema and obstructive bronchiolitis, which is small airways disease. Emphysema and obstructive bronchiolitis have varying contributions across individuals (Wright & Churg 2008). If the destruction of small airways is severe can result to formation of large air pockets referred to as bullae, which replaces the lung tissue and the condition is referred to as bullous emphysema. The development of COPD is significant to chronic inflammatory response to inhalable irritants. The inflammatory condition can be enhanced by chronic bacterial infections (Wright & Churg 2008). Inflammation is usually induced by inflammatory cells particularly neutrophil granulocytes and macrophages, which are the types of white blood cells.
Individuals who smoke are at greater risk because their inflammation additionally incorporates Tc1 lymphocyte and eosinophil. Inflammation as mediated by inflammatory cells is partly contributed by inflammatory mediators such as chemotactic factors (Wright & Churg 2008). Oxidative stress had been identified to be involved with damage of the lungs. Oxidative stress usually arises from high concentration of free radicals that are carried by tobacco components in smoke and others are produced by inflammatory cells (Chapman 2009; Jindal 2013). The activated inflammatory cells usually cause breakdown of the connective tissue by secretory proteases and due to insufficient inhibition by protease inhibitors. Note that it’s the destruction of connective tissues of the lungs that leads to development of emphysema (Murray et al. 2012). Emphysema is known to interfere with airflow, poor absorption, and release of respiratory gases. The released inflammatory mediators in the lungs have been associated with general muscle wasting that often occurs in COPD. Reduced of airflow especially when breathing out increases pressure in the chest that result to compression of airways and leads to retention of more air of the previous breathe within the lungs during the onset of the next breathing cycle (Bradley & O’Neill 2005). The resultant increased air volume in the lungs or air trapping is referred to as hyperinflation, which causes shortness of breath that is exhibited as a major symptom of COPD. The concept behind these processes and symptomatic expression is that it is difficult to breathe in when the lungs is already full occupied with air (Bradley & O’Neill 2005). Hyperinflation also causes low oxygen levels and high carbon dioxide levels in blood. The degree of hyperinflation can be enhanced during exacerbations, which causes increase in airway inflammation (Simoens et al. 2013; Barr et al. 2003). Other pathophysiological cases have been characteristic airway hyperresponsiveness to irritants, which are similar to those of asthma. COPD has also been associated with changes of lung circulatory system. For instance, emphysema leads to breakdown of capillaries in the lungs whereas low oxygen levels for prolonged period can lead to narrowing of the lung arteries (Wright & Churg 2008). The resultant effects of these changes include increased blood pressure and cor pulmonale that is partly caused by increased blood pressure.
The buffer recipies were pre-treated as follows. Citrate Buffer was prepared by dissolving 2.1 g of Tri-sodium citrate in 1000ml of Dh2O. Its pH was adjusted to 6.0 using few drops of 1M HCl. 1 mM EDTA Buffer was prepared by dissolving 0.37g of EDTA in 1000 ml of water and the PH adjusted to 8.0. Tris-EDTA Buffer was prepared by dissolving 1.21g of Tris Base and added with 0.37g of EDTA (Ethylene diamine – NNNN – tetra acetic acid di-sodium salt). The pH was adjusted to 9.0 and added with 0.5 ml Tween-20 and mixed well.
The TBS 10x was prepared using 24 g of Tris base (Formula weight: 121.1 g) and 88 g of NaCl (Formula weight: 58.4 g), which were dissolved in 900 ml of distilled water. Using the 12N HCl, the pH was adjusted to 7.6 then added with distilled water to obtain the final volume of 1 liter. This solution was used to prepare 1X solution by mixing 1 part of the 10X solution with 9 parts of distilled water. The pH was adjusted to 7.6 again and thus the final molar concentrations of the 1X solution included 20 mM for Tris and 150 mM for NaCl. The 1X TBS/ Tween solution was prepared by dissolving 500 μL of Tween-20 to 500 ml of 1X TBS. Preparation of Biotinylated secondary antibody involved adding 15 μL of normal blocking serum stock to 1 ml PBS in the mixing bottle and then adding one 5 μL of biotinylated antibody stock. Preparation of Hydrogen Peroxide (H2O2) involved mixing 48.5 ml of methol and 1.5 ml of 3% hydrogen peroxide.
Preparation of ABC Reagent involved adding exactly 2 drops of REAGENT A to 5 ml of TBS in the ABC Reagent large mixing bottle and then exactly 2 drops of REAGENT B was added to the same mixing bottle and mixed immediately. The VECTASTAIN ABC Reagent was left to settle after mixing for about 30 minutes before use. Both bottles were shaken well then added with 1 drop (approx. 30 μl) of ImmPACT DAB Chromogen concentrate to 1 ml of ImmPACT diluents and mixed well and the resultant ABC consisted of TBS + A + B.
The slides were blocked using normal serum that was obtained from the same animal that the secondary antibody was obtained in order to ensure that any non-specific or background staining from secondary antibody was blocked. The normal serum would bind to non-specific targets on the tissue or cells. The usual dilution of normal serum was set at 15 μl per 1 ml. The primary antibodies used in this research were mouse anti human and they included AE1/ AE3, CCR5, CCR7, IL-6, and IL-8. The secondary antibody was the horse anti mouse and blocking was done in horse serum (i.e. this involved the use of the mouse kit). The SA100 was the rabbit anti human and its secondary was goat anti rabbit and blocking was done in the goat serum (This involved the use of rabbit kit). The slides were blocked using the normal serum that was obtained from the same animal from which the secondary antibody was obtained.
Immunohistochemistry (IHC) of the FFPE Tissue involved standard single label immunoperoxidase. The tissue sections were de-waxed through xylene (2 x 5 minutes) then dehydrated through ethanol: 100% for 5 minutes; 90% ethanol for 3 minutes; 75% ethanol for 2 minutes; and 50% ethanol for 1 minute. Thereafter, the sections were placed under running water for 5 minutes and carefully dry slide with tissue paper while taking precaution not to wipe off the sample. The samples were pre-treated in appropriate buffer at pH 6, which involved placing the slides in a glass coplin jar. The coplin jar was placed into 1L beaker and filled with buffer and covered with cover glass and placed into the microwave that was operated at 1200w (in 116) – 20 minutes, 60% power. It was important not to place only the coplin jar in the microwave since it would explode and thus generating a risk of cuts and burns. Using heatproof gloves, the beaker/coplin jar assembly and the sections were removed from the microwave and allowed to cool in the buffer for 20 minutes. The slides were placed in running water for 5 minutes and then carefully dry slide with tissue paper while taking precaution not to touching the sample. The sections were drawn around using the PAP pen. The primary antibody was incubated in dilute normal serum for 2 hours at room temperature. On the following day, the samples were washed in TBStween 3 times for 3 minutes and incubated in vector biotinylated secondary antibody (30 minutes at room temperature). Thereafter, they were washed in TBStween (3 x 3 minutes) and blocking of the endogenous peroxidase was done using 3% H2O2 in methanol for 30 minutes at room temperature. Thereafter, the ABC was prepared in Elite Px, which was supposed to be 30 minutes before use. The samples were washed in running water for 5 minutes, in TBS for another 5 minutes, and incubated in ABC-Px solution for 30 minutes at room temperature. Thereafter, the samples were washed in TBStween 3 times for 3 minutes and incubated in DAB substrate before visualization under microscope and dH2O was used to stop the reaction.
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