Club cell protein 16 (CC16) is the most abundant protein in broncho-alveolar secretions. CC16 inherits an anti-inflammatory property in lungs. Chronic Obstructive Pulmonary disease (COPD) is caused due to exposure of lungs to smoke or any other particulate pollutants1. CC16 has a molecular mass of 16 kilo Dalton, and belongs to the secretory globin sub-family of proteins. CC16 is a homodimeric protein with identical 70-amino acid subunits linked in an antiparallel orientation by two disulfide bonds2.
The main source of CC16 is the Club cell (formerly called Clara cell) which is a non-ciliated, non-mucous secreting club-shaped cell present mainly in bronchioles as well as basal cells found in large airways. The density of Club cells throughout the respiratory tract varies substantially between species. In humans, Club cells represent 1% of all airway epithelial cells in the bronchioles and 5% in the respiratory bronchioles3. Other organs that contain CC16-secreting cells are the prostate, ovaries, pancreas, mammary glands, and uterine endometrium4, 5.
As per the literature data, many chronic pulmonary inflammatory diseases such as anthraco-silicosis, chronic obstructive pulmonary disease (COPD), asthma etc. causes depletion of CC16. COPD is a condition of lungs in which the pathogenesis is still not very clear. The main reason being progressive symmetric erythro-keratodermia develops during COPD causing hindrance in the airway. The etiological factors can be many such as breathing in of particulate dust particles, bacterial infections or smoking. In all types of cases, Club cells are degenerated and reduced in number resulting in decreased levels of CC16 in Broncho-alveolar lavage fluid (BALF) and serum. The anti-inflammatory and protective effect of CC16 on the airway epithelium is gone resulting in inflammation of lungs. In case of per-acute or acute attacks, the CC16 level increases and results in the repair of the airway epithelium. But in cases of chronic exposures, the CC16 levels reduce gradually resulting in the chronic inflammation which is ultimately leading towards fibrosis of lungs.
Silicosis is an irreversible occupational ailment of the respiratory system caused by the invasion of lung tissue (parenchyma) due to dust consisting of crystalline silica or silicon dioxide of respirable size (less than 10µ in diameter). Individuals with various exposure interval ranging from 2 to 15 years or more in the industries like mines, stone quarry, agate, construction sites and non-metallic product manufacturing units for example refractory (articles with heat resistant ability), ceramic, glass, mica, and structural clay are more prone to silicosis6. These micro-particles get trapped in the interstitial lung collagen tissue resulting in fibrosis of the lung. Therefore, it becomes one of the major occupational health hazards for the silica dust exposed workers working in relevant industries. Regrettably, most of the silicosis cases remain undiagnosed or misdiagnosed at an early stage due to asymptomatic or mild symptomatic nature of the initial stage of the disease, lack of suitable biomarker for early detection, poor health-seeking behavior of the workers and poor occupational health care delivery service at the working areas, particularly in unorganized sectors7, 8, 9.
Patients with silicosis are prone to develop pulmonary tuberculosis also called silico-tuberculosis, probably due to destruction of alveolar macrophages. Differential diagnosis is difficult unless the physician is aware of the occupational history of silica exposure, which is very subjective in nature. Also, it becomes very difficult to differentiate between silicotic nodules and tuberculous infiltration in radiography. Additionally, the difficulty in isolation of Mycobacterium tuberculosis from sputum of silico-tuberculosis patients as silicotic fibrosis prevents discharge of Mycobacterium in the sputum, making the situation more difficult10. Hence, a suitable biomarker is required for early detection of silicosis. This will augment in the prophylaxis and control of advanced silicosis and silico-tuberculosis patients.
The diagnosis of silicosis needs to be confirmed by chest radiology following clinical examination with a history of occupational exposure to silica dust for a varying period. X-ray of chest shows bi-lateral pathognomonic nodular opacities in silicosis. Diagnosis is invariably made at an advanced or end stage when it is irreversible. Moreover, silicosis patients are susceptible to tuberculosis, which is often difficult to diagnose and treat resulting in drug resistant tuberculosis. Considering above, a suitable biomarker for early detection of silicosis is needed to protect these vulnerable workers.
A number of anti-inflammatory biomarkers for early diagnosis of silicosis have been attempted, but most of these were found to be non-specific and hence conferred unsuitable for diagnosis of lung related pathologies11, 12. Club cell protein (CC16) is secreted by Club cells of Broncho-alveolar epithelial tissue of the lung13 (Bernard et al., 1994). CC16 is proposed to be a peripheral marker of respiratory epithelial injury that protects the respiratory tract against oxidative stress-induced inflammation14, 15, 16 and passively diffuses in bronchoalveolar-blood barrier to plasma17. The serum concentration of CC16 can be used to decipher the degree of respiratory tract injury and lung alveolar capillary barrier integrity at an early stage. Though the exact physiological mechanism of CC16 remains unknown, but evidences suggest significant reduction of CC16 in silica dust-exposed workers with no change in respiratory symptoms, normal chest radiology and lung function tests indicates that serum CC16 could be an early asymptomatic detection tool for silicosis among silica-exposed population at risk.
At present-day, the CC16 detection is performed with commercially available enzyme linked immunosorbent (ELISA) assays of clinical field application18 (Biovendor – Laboratorni) that requires to be imported from the foreign countries. The available commercial assays are very expensive and the cost cannot be afforded by the daily wage workers. Thus, there is a need for economical, user friendly and rapid detection devices and methods which does not require expensive instrumentation or specialized skills for testing and analysis. Considering the huge burden of silicosis patients in the country, all primary health care centers need to have some kind of users’ friendly point of care screening/diagnostic tool for early detection of silicosis so that occupational health care delivery services could be extended there. This will revolutionize the occupational health scenario of the country to a great extent focusing silicosis and silico-tuberculosis.
In the current study we describe a Point of Care assay that can be particularly employed for semi-quantitative estimation of CC16 in human serum samples. This assay can be used periodically at regular intervals to assess the serum CC16 levels among workers with history of silica dust exposure. This assay would give an idea about an estimated lung injury caused by silica dust exposure before advising for their radiological confirmation to arrive at a conformed diagnosis. So, it may be considered as a proxy bio-marker and screening tool for silicosis.