Measuring Carbon Content in Airway Macrophages Exposed to Carbon-Containing Particulate Matters
Summary
This article describes a detailed experimental protocol for measuring the carbon content of airway macrophages with the aim of assessing the internal exposure dose at the level of individual particulate matter exposure.
Abstract
Pulmonary macrophages exhibit a dose-dependent pattern in phagocytizing particles. Following engulfment, these macrophages are subsequently excreted with sputum, rendering macrophages and particles visible and quantifiable under light microscopy. Notably, elemental carbon within the mammalian body originates exclusively from external contaminants. Consequently, the carbon content in airway macrophages (CCAM) serves as a valid exposure biomarker, accurately estimating individual exposure to carbon-containing particulate matter (PM). This article delineates a protocol involving sputum collection, preservation, processing, slide preparation, and staining, as well as macrophage photo acquisition and analysis. After removing the macrophage nuclei, the proportion of cytoplasm area occupied by carbon particles (PCOC) was calculated to quantify carbon content in each macrophage. The results indicate an elevation in CCAM levels after exposure to carbon-containing PM. In summary, this non-invasive, precise, reliable, and standardized method enables the direct measurement of carbon particles within target cells and is utilized for large-scale quantification of individual CCAM through induced sputum.
Introduction
Ambient air pollution is associated with deaths due to respiratory and cardiovascular diseases, posing a serious threat to human health1,2. Epidemiological data indicate that chronic exposure to ambient particulate matter less than or equal to 2.5 µm in diameter (PM2.5) is responsible for the premature deaths of between 4 and 9 million people globally. PM2.5 was ranked as the fifth most important risk factor for global mortality in the 2015 Global Burden of Disease, Injuries, and Risk Factors Study (GBD)3,4,5,6. Studies have found that adherence to WHO air pollution guidelines could prevent 51,213 deaths per year from PM2.5 exposure3. Currently, most studies lack the evaluation of intra-individual exposure and are only based on crude evaluations at larger regional monitoring sites, which are far from individual exposure levels. The available biomarkers of internal exposure, such as urinary PAHs and benzo(a)pyrene, do not reflect the associations between particulate matter exposures and health effects7,8,9. This leads to the inability to establish an accurate relationship with health effects. Therefore, the search for markers that reflect the level of particulate matter exposure in an individual is one of the keys to accurate exposure assessment for individuals.
Particles inhaled into the bronchi can be excreted with sputum through ciliary oscillations in the bronchi. The lack of a ciliated mucous flow transport system in the alveoli means that the primary clearance route for particles entering the alveoli is through phagocytosis and translocation by macrophages10,11. Based on the anatomical structure of the lung, its clearance of insoluble particulate foreign matter is slow. This allows particulate matter to interact with lung cells for extended periods and initiate various biological effects, causing damage to lung tissue and other organs12,13. Stimulation by particulate matter leads to macrophage activation, triggering a cascade of inflammatory factors in the lungs that can cause a systemic inflammatory response14. Considering the crucial role of macrophage cytophagy in eliciting cytokine storms in the lungs, it is theorized that carbon particles from lung macrophages could reflect the biologically effective dose of airborne carbon-nucleated particulate exposure15. Furthermore, since there is no aggregation of elemental carbon in mammalian cells and carbon-containing particles can be observed as black particulate matter under a light microscope, the collection of alveolar and bronchial macrophages and measurement of the carbon content in them can serve as a marker for evaluating particulate matter exposure16.
This study identified a method for accurately assessing individual particulate matter exposure levels, known as the Carbon Content of Airway Macrophages (CCAM). Specifically, population sputum samples were collected after participants inhaled hypertonic saline generated by an ultrasonic nebulizer. These samples were then preserved using a fixative solution. Airway macrophages were isolated, stained, and photographed under a light microscope to identify macrophages containing carbon-containing particles, which were then quantified. This method provides a biological marker for accurately assessing individual particulate matter exposure levels. It establishes a methodological foundation for investigating the relationship between particulate matter exposure and health effects, serving as a research basis for exploring associations between PM exposure and health outcomes such as lung diseases.
Protocol
Representative Results
Discussion
This study presents a detailed experimental protocol for using induced sputum-derived CCAM as a biological marker for internal exposure to atmospheric particulate matter. CCAM can be detected and quantified through optical microscopy, serving as a precise internal exposure biomarker reflecting the relationship with health effects. Therefore, there is a need to establish and optimize a convenient, reliable, and efficient induced sputum preservation method and a CCAM quantification method to standardize the methodological …
Disclosures
The authors have nothing to disclose.
Acknowledgements
This work was financially supported by the National Natural Science Foundation of China (82273669, 82241086, 42207488), the Taishan Scholars Program of Shandong Province (No. tsqn202211121), and the Innovation and Technology Program for the Excellent Youth Scholars of Higher Education of Shandong Province (2022KJ295).
Materials
| 3 mL sterile straws | Shanghai YEASEN Biotechnology Co., LTD | 84202ES03 | |
| 50 mL centrifuge tube | Thermo Fisher Scientific, USA | 339652 | |
| Absolute ethyl alcohol | Sinopharm Group Chemical Reagent Co. LTD | 64-17-5 | |
| Cedar oil | Shanghai McLean Biochemical Technology Co., LTD | C805296 | |
| Diff-quick staining solution | Shanghai YEASEN Biotechnology Co., LTD | 40748ES76 | |
| Dithiothreitol | Solebo Bio Co., LTD | D8220 | |
| Duchenne phosphate buffer (DPBS) | Thermo Fisher Scientific, USA | 14190144 | |
| Microscope camera | Olympus Corporation of Japan | DP72 | |
| Neutral tree gum | Solebo Bio Co., LTD | G8590 | |
| Nylon filter membrane 70um | BD Falcon Bioscience, USA | 211755 | |
| Optical microscope | Olympus Corporation of Japan | BX60 | |
| Polyethylene glycol | Sinopharm Group Chemical Reagent Co. LTD | 25322-68-3 | |
| Ultracentrifuge | Thermo Fisher Scientific, USA | SL40R | |
| Viscous slide | Jiangsu SHitAI EXPERIMENTAL Equipment Co. LTD | 188105 |
References
- Münzel, T., et al. Environmental stressors and cardio-metabolic disease: part I-epidemiologic evidence supporting a role for noise and air pollution and effects of mitigation strategies. Eur Heart J. 38 (8), 550-556 (2017).
- Guarnieri, M., et al. Lung function in rural Guatemalan women before and after a chimney stove intervention to reduce wood smoke exposure: results from the randomized exposure study of pollution indoors and respiratory effects and chronic respiratory effects of early childhood exposure to respirable particulate matter study. Chest. 148 (5), 1184-1192 (2015).
- Khomenko, S., et al. Premature mortality due to air pollution in European cities: a health impact assessment. Lancet Planet Health. 5 (3), e121-e134 (2021).
- Kulkarni, N., Pierse, N., Rushton, L., Grigg, J. Carbon in airway macrophages and lung function in children. N Engl J Med. 355 (1), 21-30 (2006).
- Krzyzanowski, M. WHO air quality guidelines for Europe. J Toxicol Environ Health A. 71 (1), 47-50 (2008).
- Cohen, A. J., et al. Estimates and 25-year trends of the global burden of disease attributable to ambient air pollution: An analysis of data from the Global Burden of Diseases Study 2015. Lancet. 389 (10082), 1907-1918 (2017).
- Hajat, A., et al. The association between long-term air pollution and urinary catecholamines: Evidence from the multi-ethnic study of atherosclerosis. Environ Health Perspect. 127 (5), 57007 (2019).
- . Traffic-related air pollution: A critical review of the literature on emissions, exposure, and health effects Available from: https://www.healtheffects.org/publication/traffic-related-air-pollution-critical-review-literature-emissions-exposure-and-health (2010)
- Uccelli, R., et al. Female lung cancer mortality and long-term exposure to particulate matter in Italy. Eur J Public Healt.h. 27 (1), 178-183 (2017).
- Zhang, L., et al. Indoor particulate matter in urban households: sources, pathways, characteristics, health effects, and exposure mitigation. Int J Environ Res Public Health. 18 (21), 11055 (2021).
- Wang, K., Zeng, R. Cough and expectoration. Handbook of Clinical Diagnostics. , 27-29 (2020).
- Qi, Y., et al. Passage of exogeneous fine particles from the lung into the brain in humans and animals. PNAS. 119 (26), e2117083119 (2022).
- Comunian, S., Dongo, D., Milani, C., Palestini, P. Air pollution and COVID-19: The role of particulate matter in the spread and increase of COVID-19’s morbidity and mortality. Int J Environ Res Public Health. 17 (12), 4487 (2020).
- Sharma, J., et al. Emerging role of mitochondria in airborne particulate matter-induced immunotoxicity. Environ Pollut. 270, 116242 (2021).
- Uribe-Querol, E., Rosales, C. Phagocytosis: Our current understanding of a universal biological process. Front Immunol. 11, 1066 (2020).
- Kupiainen, K., Klimont, Z. Primary emissions of fine carbonaceous particles in Europe. Atmos Environ. 41 (10), 2156-2170 (2007).
- Tueller, C., et al. Value of smear and PCR in bronchoalveolar lavage fluid in culture positive pulmonary tuberculosis. Eur Respir J. 26 (5), 767-772 (2005).
- Takiguchi, H., et al. Macrophages with reduced expressions of classical M1 and M2 surface markers in human bronchoalveolar lavage fluid exhibit pro-inflammatory gene signatures. Sci Rep. 11 (1), 8282 (2021).
- Andelid, K., et al. Lung macrophages drive mucus production and steroid-resistant inflammation in chronic bronchitis. Resp Res. 22 (1), 172 (2021).
- Weiszhar, Z., Horvath, I. Induced sputum analysis: Step by step. Eur Respiratory Soc. 9, 300-306 (2013).
- Popov, T., et al. Some technical factors influencing the induction of sputum for cell analysis. Eur Respir J. 8 (4), 559-565 (1995).
- Sumner, H., et al. Predictors of objective cough frequency in chronic obstructive pulmonary disease. Am J Respir Crit Care Med. 187 (9), 943-949 (2013).
- Brugha, R. E., et al. Carbon in airway macrophages from children with asthma. Thorax. 69 (7), 654-659 (2014).
- Simpson, J. L., Timmins, N. L., Fakes, K., Talbot, P. I., Gibson, P. G. Effect of saliva contamination on induced sputum cell counts, IL-8 and eosinophil cationic protein levels. Eur Respir J. 23 (5), 759-762 (2004).
- Risse, E. K., Van’t Hof, M. A., Laurini, R. N., Vooijs, P. G. Sputum cytology by the Saccomanno method in diagnosing lung malignancy. Diagn Cytopathol. 1 (4), 286-291 (1985).
- Eells, T. P., Pratt, D. S., Coppolo, D. P., Alpern, H. D., May, J. J. An improved method of cell recovery following bronchial brushing. Chest. 93 (4), 727-729 (1988).
- Bai, Y., et al. Mitochondrial DNA content in blood and carbon load in airway macrophages. A panel study in elderly subjects. Environ Int. 119, 47-53 (2018).
- Jary, H., Rylance, J., Patel, L., Gordon, S. B., Mortimer, K. Comparison of methods for the analysis of airway macrophage particulate load from induced sputum, a potential biomarker of air pollution exposure. BMC Pulm Med. 15, 1-9 (2015).
- Gilbert, D. F., Meinhof, T., Pepperkok, R., Runz, H. DetecTiff: A novel image analysis routine for high-content screening microscopy. J Biomol Screen. 14 (8), 944-955 (2009).
- Dai, Y., et al. Effects of occupational exposure to carbon black on peripheral white blood cell counts and lymphocyte subsets. Environ Mol Mutagen. 57 (8), 615-622 (2016).
- Cheng, W., et al. Carbon content in airway macrophages and genomic instability in Chinese carbon black packers. Arch Toxicol. 94 (3), 761-771 (2020).
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