Protease Regulation in COPD: An Immunohistochemical Analysis of COPD Tissue
The risk of developing Chronic Obstructive Pulmonary Disease (COPD) is higher in heavy smokers. COPD is characterized by poorly reversible lung function failure. The process of inflammatory cell recruitment is contributed by smoke-induced inflammation involving neutrophils and macrophages, which have the ability to release proteolytic enzymes. These enzymes are known to fight lungs pathogens. The production and secretion of these enzymes is carefully regulated by inhibitors such as down-regulation when not needed. In COPD cases, this enzyme appears to be dysregulated. This research utilized immunohistochemical (IHC) techniques to characterize the level of expression of protease and inhibitor in paraffin that has been embedded with lung tissue resections obtaine from patients with respiratory disease. The analysis of the findings involved the determination of correlation of protease and inhibitor expression and in relation to morphological appearance of lung resections and patient demographics that included lung function and smoking pack year history.
Chronic Obstructive Pulmonary Disease (COPD) is also referred to as Chronic Obstructive Lung Disease (COLD) or Chronic Obstructive Airway Disease (COAD). COPD is a type of obstructive lung disease that is characterized by progressive and chronically poor airflow that makes it hard to breath. If accompanied with coughing, the patient produces large quantities of mucus. The disease is also characterized by wheezing, shortness of breath, and chest tightness among other symptoms. Cigarette smoking has been identified as the leading cause of COPD. Other etiological factors or major contributors of COPD include air pollution, chemical fumes, and dust among others. In order to understand COPD, it would be better to understand the functional principles of the lung. The lung is an organ that is specialized for respiration through breathing. The air that is breathe moves into the lung through the windpipe (bronchial) that divides into thousands of smaller tubes and tubules referred to as bronchioles within the lungs. The terminals of these tubules are the air sacs referred to as alveoli, which are bunches of tiny rounded air sacs. The alveoli are surrounded by capillaries (small blood vessels), which run through the surface of the alveoli. When the air that is breathed reaches the alveoli, the oxygen crosses through the walls of the alveoli and the capillaries into the blood in the lumen of the capillaries. Simultaneously, the carbon dioxide (waste gas) carried in the blood moves in the opposite direction i.e. from the capillaries into the alveoli from which it is excreted through breathing out. An individual with COPD has a characteristic less air flow in and out of the airways because of one or more of the following features. First, the alveoli and airways lose their elasticity. Second, the individual exhibit destroyed walls between multiple alveoli. The destruction of the alveoli wall can result to formation of fewer large alveoli. Third, the patient has inflamed or thickened wall of the airways. Fourth, the airways produce more mucus than usual, which can result to clogging.
There are two main conditions that are associated with COPD and they include emphysema and chronic bronchitis. The emphysema condition is characterized by damaging of the walls between many of the air sacs, which results to the loss of shape of alveoli that become floppy. It also results to destruction of the alveoli walls leading to fewer and large air sacs instead of normal many and tiny alveoli. This in turn results to reduced amount or rate of gas exchange in the lungs. On the other hand, the chronic bronchitis condition is characterized irritated and inflamed lining of the airways resulting to thickening of the lining. The condition is also associated with production of thick mucus in the airways that makes it hard to breathe. Many people with COPD have both conditions – emphysema and chronic bronchitis. In this regard, the occurrence of either condition or both is referred by the general term COPD.
COPD has been identified as a major cause of disability and one of the leading contributors of mortality. The disease has been associated with high incidence and prevalence rates in many countries across the globe. It has also been established through research that many people may be having the disease but do not know about its development and progression. The development of COPD is usually spontaneous and thus symptoms usually worsen with time, which results to reduced ability to perform routine activities. The progression of the disease to severe stages can prevent the patient from performing even the basic activities such as cooking, walking, and taking care of other personal needs. According to hospital records, most of the positive COPD diagnosis usually occur in middle-age, older adults, and the aged. The disease is non-communicable and thus cannot be transmitted from person to person. To date, there is no cure of COPD yet and there is also no medical/ therapeutic mechanism for reversing the damage caused to the airways and lungs. The only existing best management mechanisms involves lifestyle changes, which makes the patient feel better, increase activity, and slows the progression of the disease.
Smoking is the leading cause of COPD because the inhalation of cigarette smoke causes irritation and inflammation of the lungs resulting to scarring. Consistent smoking leads to permanent changes in the lungs resulting to thickening of the airways and increased production of mucus. These processes are associated with damaging of the delicate walls of the alveoli leading to emplysema and the lungs lose its elasticity. Additionally, the condition also causes the smaller airways to scar and thus inflame to become narrower. These changes results to the commonest symptoms of COPD that include breathlessness, coughing, and phlegm. Apart from cigarette smoking, inhalation of fumes and particles/ dust, air pollution, and genetic disorders have also been associated with development and progression of COPD but the incidences are rare. These rare etiologies have been associated with inhalation of secondhand smoke and other related agents from the environment particularly from the work place (occupational) hazards. In the case of genetic etiologies, a condition referred to as alpha-1 antitrypsin deficiency has been associated with COPD. People with this condition have been identified to having low level of alpha-1 antitrypsin (AAT), which is a protein that is synthesized in the liver. Individuals with low level of AAT proteins are at risk of developing lung damage that can result to irritation and inflammation, which are the initial inducing events of developing COPD. The progression of COPD can occur very quickly if the etiological factors interact with the lung high at a increased rate. For instance, smoking and inhalation of other environmental –based lung irritants such as fumes can worsen the progression of COPD. It should be noted that COPD is quite uncommon in individuals with asthma though some people with this condition can develop COPD. Asthma is known to be a chronic lung condition that causes inflammation and narrowing of the airways and successful treatment usually reverse this inflammation and narrowing. In case an individual with asthma does not seek appropriate medication then COPD can develop.
Pre-treatment of the Buffer Recipies
- Citrate Buffer
The preparation of the citrate buffer involved 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
The 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
Tris-EDTA buffer was prepared by dissolving 1.21g of Tris Base (C4H11NO3, C10N14N2Na2O8.2H2O). It was 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.
Preparation of TBS 10X (Concentrated Tris-Buffered Saline) – 1 Liter
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) that were dissolved in 900 ml of distilled water. The pH was adjusted to 7.6 using 12N of HCl. It was then added with distilled water to obtain the final volume of 1 liter. From this solution, 1X solution was prepared by mixing 1 part of the 10X solution with 9 parts of distilled water and the pH was adjusted to 7.6 again. In this regard, the final molar concentrations of the 1X solution were 20 mM Tris and 150 mM NaCl.
Preparation of 1X TBS/ Tween
This solution was prepared by dissolving 500 μL of Tween-20 to 500 ml of 1X TBS.
Preparation of Biotinylated Secondary Antibody
This 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)
This involved mixing 48.5 ml of methol and 1.5 ml of hydrogen peroxide (3%).
Preparation of ABC Reagent
This involved the addition of exactly 2 drops of REAGENT A to 5 ml of TBS in the ABC Reagent large mixing bottle. Thereafter, exactly 2 drops of REAGENT B was added to the same mixing bottle and mixed immediately. The VECTASTAIN ABC Reagent was allowed to stand 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. Note that ABC consisted of TBS + A + B.
The slides were blocked using normal serum. The normal saline was obtained from the same animal in which the secondary antibody was obtained. This ensured 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
This process involved standard single label immunoperoxidase and was accomplished within 2 days.
The activities of the first day included the following. The sections were de-waxed through xylene (2 x 5 minutes). Thereafter, the sections were dehydrated through ethanol: 100% for 5 minutes; 90% ethanol for 3 minutes; 75% ethanol for 2 minutes; and 50% ethanol for 1 minute. They were then placed under running water for 5 minutes and carefully dry slide with tissue paper (It was important not to wipe off the sample). The samples were pre-treated in appropriate buffer at pH 6. Pre-treatment involved placing the slides in a glass coplin jar. The coplin jar was placed into 1L beaker and filled with buffer. The beaker was 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. With the use of heatproof gloves, the beaker/coplin jar assembly and the sections were 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 without touching the sample. The sections were drawn around using the PAP pen. This process involved the use of 100 ml of diluted antibody and blocked in vector normal serum (30 minutes at RT). The primary antibody was incubated in dilute normal serum for 2 hours at room temperature.
The activities of the second day involved the following. The samples were washed in TBStween (3 x 3 minutes). It was incubated in vector biotinylated secondary antibody (30 minutes at room temperature). Thereafter, it was washed in TBStween (3 x 3 minutes) and the endogenous peroxidase was blocked by incubating in 3% H2O2 in methanol for 30 minutes at room temperature. ABC was prepared in Elite Px 30 minutes before use. The sample was washed in running water for 5 minutes and 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 x 3 minutes) and incubated in DAB substrate before visualization under microscope and dH2O was used to stop the reaction.