Merits and demerits of various biomarkers of chronic lung diseases with special reference to club cell protein 16 (CC-16) and early detection of chronic silicosis

Silicon:

Hongli et.al. [12] used animal and human studies to explore whether silicon can be an important exposure marker in the development of silicosis. Serum levels of silicon were found to increase immediately in rats exposed to silicon dust. Similarly, their population study revealed that the silicon level in the silica exposed groups with and without obvious symptoms were significantly increased over that of the control group. In subjects with extended exposure to silica, the serum and urine silicon level in exposed workers appeared to increase rapidly, reaching its peak in 1-5 years, followed by a gradual decline thereafter. The workers with a total exposure time to silica dust of less than 10 years were further categorised into groups of 2 year intervals each. The levels of silicon in their urine and serum were found to be remarkably higher than that of the control group. The study concluded with the suggestion that determining the level of silicon in vivo might be an effective exposure marker in the diagnosis and pathogenesis of silicosis. But considering the fact that silicon level reaches a peak value between 1 to 5 year following exposure and then declines gradually, questions about its suitability for use in periodic screening.

Respirable crystalline silica:

According to Elizabeth et. al. [13], mentioned about detection of respirable crystalline silica in the exhaled breath condensate (EBC) of sandstone quarry workers, may be used as a method for detection of silicosis in them. An EBC sample predominantly contains water vapour and epithelial lung lining fluid, and is usually collected through a mouthpiece. However, despite the non-invasive nature of collection and the utility of EBC as a biological “matrix” for monitoring occupational exposure to silica dust; it provides little information about the extent of lung damage and its correlation. Also, a need for standardisation of the collection and analysis procedure is also felt. This warrants the need for further in-depth studies to establish its viability as a sensitive and specific diagnostic/screening tool for evaluating progression of silicosis [14].

Other Markers:

Xue et. al. [15] also identified other serum biomarkers for lung damage like Krebs von den Lungen-6 (KL-6), matrix metalloproteinase-2 (MMP-2), and surfactant protein D (SP-D); for diagnosis of pneumoconiosis like asbestosis and silicosis. In this study, the subjects belonged to either of the four groups: subjects with silicosis, subjects with asbestosis, dust exposed workers and healthy controls. KL-6, also known as MUC1 mucin in humans, has been used as a significant marker in diagnosis of various chronic lung fibroses. Secreted by type II pneumocytes in the alveoli of lungs, higher serum concentrations of KL-6 were linked with asbestosis, silicosis and idiopathic pulmonary fibrosis [15,16]. SP-D is one of the proteins present in the pulmonary surfactant that is also derived from type II pneumocytes. Patients with asbestosis and silicosis were found to have higher serum concentration of SP-D. However, SP-D was found to be a more effective marker for asbestosis than for silicosis. In case of MMP-2, although evidence exists that higher serum concentrations of MMP-2 are found in patients with idiopathic lung fibrosis, a strong correlation between serum MMP-2 concentration and pneumoconiosis like silicosis and asbestosis, was not established [15]. To summarise however, the authors recommended measurement of serum concentrations of all the three above-mentioned biomarkers for reliable results, and clarified that the dynamic changes in the serum concentrations of these markers could not be monitored in all the subjects [15]. Considering lack of requisite criteria for a near ideal screening/diagnostic test, there is need for further in-depth studies to understand the exact role of these biomarkers towards suitability for screening tests for silicosis and asbestosis.

Inflammatory biomarkers:

Research has shown that serum inflammatory biomarker levels are elevated in silicotic patients, but the extent of their specificity for evaluating silicosis has not been well established. A review by Gulumian et. al. [17], mentioned that even today, the clinical detection of silicosis is largely dependent on chest X ray and lung function test anomalies, both of which are advanced manifestations of the disease. They conducted a robust review of literature on available biomarkers with the aim of identifying an ideal biological marker that is capable of detecting silicosis and coal workers’ pneumoconiosis (CWP) in its early stages. These included club cell protein-16 (CC16), interleukin-8 (I L-8), platelet-derived growth factor (PDGF), 8-isoprostane, reactive oxygen species (ROS), glutathione, glutathione S-transferase activity and glutathione peroxidase (GPx) activity. Oxidative stress is a major consequence of exposure to silica, and occurs by a number of mechanisms. Biomarkers of oxidative stress, like ROS, total antioxidant level, 8-isoprostane, glutathione, GPx and Glutathione S-transferase; all carry the potential to be used as a biomarker for the early detection of silicosis and CWP. However, the non-specificity of these markers to silicosis; especially due to the fact that mineral particles other than silica may also influence the levels of these markers, have led us to explore other biomarkers. Growth factors have also been known to be involved in fibrous lung diseases. PDGF secretion by alveolar macrophages was found to be stimulated by silica dust and coal dust. However, higher than normal serum PDGF levels were only detected in advanced silicosis patients, thus using it as a biomarker for early detection seems difficult. In conclusion, the authors opined that further studies were required to analyse the validity and practical feasibility of all the above mentioned biomarkers.

TNF-alpha:

Slavov et. al. [18] conducted a study in Bulgaria, which explored the potential of tumour necrosis factor-alpha (TNF-α) as a biomarker for silicosis. They observed that TNF-α levels were similar in healthy individuals exposed to silica dust and silicosis patients. In contrast, the TNF-α levels of healthy individuals without prior exposure to silica dust were significantly lower. They also noted that there were no significant differences in the serum TNF-α levels in silicosis patients with moderate lung damage and severe lung damage. The need to correlate TNF-α levels with other biomarkers to indicate disease progression; coupled with the fact that TNF-α seemed to primarily indicate exposure to silica dust and not the extent of lung damage in silicosis patients, led us to question its feasibility as a reliable biomarker that could serve as a screening tool for silicosis.

Another longitudinal study by José et. al. [19] analysed numerous inflammatory biomarkers in silicotic patients. Manifestation of an inflammatory response to RCS, via an elevated number of lymphocytes, neutrophils and macrophages is a natural consequence. The study inferred about inflammatory markers, particularly interleukin 8 (IL-8) has the reasonable potential as a biomarker for assessing silicosis. However, it's role in the development and progression of silicosis was not well understood, and it seemed to be related to the development of progressive massive fibrosis. IL-8 is a chemotactic cytokine that can be produced by numerous tissues, including endothelial cells, macrophages and airway smooth muscle cells etc. [20]. Given its secretion by diverse tissues, it's specificity as a biomarker for silicosis or other pneumoconiosis remains uncertain.

Marker secreted predominantly from the lungs:

Club cell protein 16 or CC16 appears to be an important marker towards this. CC16 (previously known as CC10), belongs to the secretoglobin family of proteins. Distal human lung epithelium is lined with abundant club cells, which account for as many as 22% of the cells in the lung epithelium [21]. Apart from club cells lining the human lung epithelium, CC16 is produced in small amounts by other tissues in the urogenital tract. However, the serum concentration of CC16 is found to be predominantly indicative of the amount secreted by the club cells lining the lung epithelium [22]. CC16 is highly soluble and diffuses easily into the circulation, where it can be measured. Acute exposures to endotoxin and wood smoke in healthy volunteers increases serum CC16 concentrations as early as 6 hr. after exposure, returning to normal within 24 hr [23]. Bernard A et al., observed that the highest concentrations of CC16 are observed in sputum and broncho-alveolar lavage fluid, reflecting intense secretion of the protein in the airways [24]. The protein is also found in smaller concentrations, in other fluids like amniotic fluid, urine and also, semen. Broeckaert and Bernard [25] found that Club cells are one of the most multifunctional and heterogeneous cell types in the mammalian lung, with their main function being protection of the respiratory tract. CC16 concentration in broncho-alveolar lavage fluid, which correlates well with its serum concentration, has also been shown to be a viable indicator of extent of lung damage in other lung diseases like chronic obstructive pulmonary disease (COPD) and asthma. [22,26] Serum concentration of CC16 was also found to be weakly yet significantly correlated with declining lung function in COPD patients. This correlation was found to be stronger than that of other conventional inflammatory markers like IL-8 [27]. Lucky et. al. [28] mentions two mechanisms by which CC16 concentration decreases in silicotic patients. Firstly, the silica particles directly damage the Clara or Club cells, which are responsible for secretion of CC16. Secondly, these particles trigger an inflammatory response, and the Club cells are damaged by activated macrophages which release cytotoxic mediators. The authors, Jai Krishna Pandey & Deepa Agarwal reviewed the merits & demerits of various possible biomarkers of silicosis based on literature search & critical analysis [29]. They opined in their review paper that serum CC16 is a sensitive bio-marker and may be useful for silicosis detection compared to others.

Bernard AM et. al. [30] measured the concentration of CC16 in the serum of 86 miners exposed to silica and 86 control subjects matched for age, body mass index and smoking status (each group had 60 current smokers and 26 lifelong smokers). Workers were exposed to silica-rich dust in a quarry for 15.2 months on an average. Pertaining to respiratory symptoms, chest radiographs or lung function tests; no appreciable difference was detected between the exposed and control workers. However, the serum CC16 concentration was markedly decreased in silica-exposed workers (with geometric mean 12.3 μg/l) compared to that of controls (16.3 μg/l). Tobacco smoking was found to reduce serum CC16 level, which was additionally lowered following exposure to silica. The investigators concluded that serum concentrations of CC16 probably reflect the very early toxic effects of silica particles on the respiratory epithelium. This reinforces the view that serum CC16 is a sensitive marker, which might improve the ability to detect exposure to chemicals potentially harmful to the respiratory tract.

A comprehensive review for biomarkers in silicosis and CWP by Gulumian, P. J. A. Borm, V. Vallyathan, V. Castranova, K. Donaldson, G. Nelson and J. Murray [17] reinforced the fact that CC16 could be used as a “reliable marker”, even for detecting the early stages of silicosis. An Australia based research group [14] also reviewed the available literature on silicosis and opined in favour of a bio-marker for early detection of silicosis. They also reviewed earlier work on CC16 [11] by ICMR-National Institute of Occupational Health, India, and opined CC16 as a potential early marker candidate for silicosis.

Screening for silicosis using CC-16:

Recently Indian Council of Medical Research -National Institute of Occupational Health (ICMR-NIOH), has conclusively evidenced through their two research studies, that club cell protein 16 or CC16 may be a useful screening tool for early detection of silicosis among the workers with exposure to silica dust [11,31]. It may be noted that other research works on silica dust induced lung injury also supports the view of ICMR-NIOH's work [29]. CC16, being a marker of chronic lung disease due to any cause, may be used as a proxy marker for early detection of any chronic lung disease including silicosis. When CC16 is selectively used as a screening tool for screening workers with a history of silica dust exposure, it appears to be an effective marker with high sensitivity and specificity. The ICMR-NIOH's study [31] made a unique attempt to estimate and approximately quantify lung damage among silica dust exposed workers. This was done by using a novel scoring method of Lung Damage Score (LDS) using chest x-ray based opacities and involving ILO guidelines for assessing silicosis [32]. The study conclusively evidenced that serum CC16 levels are inversely related to the extent of lung damage -greater the lung damage, lower is the serum CC16 level (Figure -1).

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FIGURE-1:

CORROBORATION OF SERUM CC-16 WITH SILICOTIC LUNG DAMAGE BASED ON CHEST RADIOGRAPHY

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Smoking has been found to reduce the serum CC16 levels additionally in both healthy as well as silica dust exposed workers, as revealed in the ICMR-NIOH study [11]. Hence, the workers need to be advised to refrain from smoking for 1-2 months prior to the screening, which would repair/regenerate the damaged cells and bring back CC16 level close to their pre-smoke level [11,31]. Alternatively, if refraining from smoking is not feasible, some adjustments to serum CC16 levels may be made. In case of heavy smokers (>10 cigarettes per day), 2 ng/ml and for moderate smokers (5 to <10 cigarettes per day), 1 ng/ml may be added to their serum CC16 level to adjust for the declined CC16 level due to smoking [11]. The inverse relationship of serum CC16 with progressive silica induced lung injury appears to be at least one step forward towards finding out a suitable biomarker for assessing chronic lung disease such as silicosis. This may be justified in absence of other suitable mechanisms for early detection of silicosis.