Measuring Carbon Content in Airway Macrophages Exposed to Carbon-Containing Particulate Matters

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 health^1,2. 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 effects^7,8,9. 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 macrophages^10,11. 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 organs^12,13. 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

The study received approval from the Medical Ethics Committee of the Institute of Occupational Health and Poison Control, Chinese Center for Disease Control and Prevention (NIOHP201604), with written informed consent obtained from all subjects prior to the study and biological sample collection. For this study, carbon black packers who had been working in a carbon black factory for more than 1 year and were exposed to carbon black aerosols were selected. Waterworks workers in a waterworks factory with no significant occupational exposure to harmful factors were recruited from the local area as a control population to establish the study. The following inclusion and exclusion criteria were selected for the study population: Inclusion criteria for carbon black packers included direct contact with carbon black during most shifts and a minimum of one year's work in the carbon black exposure area. Waterworks workers were included if they had no occupational exposure to carbon black or other pollutants in their work environment. Exclusion criteria encompassed workers with chronic diseases such as cancer, those who had undergone X-ray exposure in the past three months, individuals with a history of pulmonary tuberculosis, pulmonary surgery, viral myocarditis, congenital heart disease, recent fever, or inflammation, and workers who had taken antibiotics recently. The reagents and the equipment used in the study are listed in the Table of Materials.

1. Sputum preservation

1. Prepare the fixative solution following the steps below:
   1. Melt 2% polyethylene glycol (PEG1500) in a water bath. Take 20 mL of melted PEG1500 in a centrifuge tube. Add 20 mL of 50% ethanol to the centrifuge tube and shake the mixture thoroughly to create the mother liquor.
   2. Gradually pour 40 mL of the mother liquor into 960 mL of 50% ethanol. Shake the solution well to form the Saccomanno fixation solution. Store the solution in a cool, dark place.
2. Add 20-30 mL of the prepared fixative solution to a centrifuge tube containing approximately 2 mL of sputum. Mix the contents thoroughly.
   NOTE: Ensure the total volume does not exceed 40 mL.
3. Seal the centrifuge tube with a sealing film. Store it in a cool, dark place. Transport the tube to the laboratory for further processing when ready.
   NOTE: Always melt PEG1500 in a water bath and use as per the prescribed dosage.

2. Preparation of cell suspension in sputum

1. Prepare the digestive solution by dissolving 0.1 g of dithiothreitol in 100 mL of saline as per the actual dosage. Store the prepared solution at 4 °C for up to 48 h.
2. Centrifuge the tube containing the sputum at 1,998 x g for 30 min at 4 °C.
3. Discard the supernatant. Add an equal volume of sputum digest to the precipitate. Vortex and shake the mixture thoroughly.
   1. Place the tube in a 37 °C water bath until complete liquefaction (10-15 min).
      NOTE: Continuously shake the tube during the liquefaction process.
4. Filter the liquefied mixture with a 70 µm filter membrane.
5. Centrifuge the filtered solution at 500 x g for 7 min at 4 °C.
6. Discard the supernatant, retaining the cell precipitate.
7. Resuspend the cell precipitate in Duchenne phosphate buffer. Centrifuge the resuspended solution at 500 x g for 7 min at 4 °C to obtain a pure cell precipitate.

3. Preparation of cell smear

1. Add 200-600 µL of phosphate buffer to the cell sediment. Mix the contents thoroughly; adjust the buffer volume based on cell counting results (aim for >1.0 × 10⁵ cells).
2. Take 20 µL of the cell suspension and create a cell smear using the hematocrit method¹⁷.
3. Let the smear air-dry naturally (within 48 h). Fix the smear with a commercially available staining solution for 10 s.
4. Immerse the smear in A staining solution for 8 s. During staining, gently lift the slide up and down, and rinse off excess stain with running water.
5. Stain the smear in B staining solution for 8 s. Lift the slide during staining and rinse off excess stain under running water.
   NOTE: Staining solutions A and B are available in the commercially available staining kit.
6. Dip the slide in anhydrous ethanol twice, each time for 2-3 s.
7. Once dry, apply an appropriate amount of neutral gum. Seal the slide with coverslips.

4. Quantitative analysis of CCAM

1. Use a light microscope with a 100x oil lens objective. Capture images of randomly selected, well-stained, and morphologically intact macrophages. Take 50 macrophage pictures randomly for each sample.
2. Perform image analysis in the Image J software following the steps below:
   1. Measure the scale and determine the actual length and pixel-to-pixel conversion (282 pixels = 10 µm). Set the scale in Image J (Analyze > Set scale), input Distance in pixels, actual length, unit of length (µm), and tick Global.
   2. Use an irregular shape to outline the cells. Remove the background (freehand selections, Edit > Clear outside). Measure the total area of the cells (Analyze > Measurement).
   3. Cut out the nucleus (Edit > Cut). Convert the grayscale image to black and white (Image > Type > 8 bit).
   4. Adjust the grayscale value specific to each cell staining for accurate carbon particle counting (Image > Adjust > Threshold > Apply, Analyze >> Measurement).
   5. Calculate the carbon content of 50 macrophages per sample based on the measured area and image analysis in Image J software.

Representative Results

The sputum, preserved and processed with the fixative solution, displayed intact macrophage morphology under an optical microscope during morphological examination. The macrophages exhibited clear, round, or kidney-shaped, easily stainable cell nuclei. Following staining, the cell nuclei appeared bluish-purple, while the cytoplasm was light pink or light blue. The microscopic field showed minimal impurities, which facilitated easy cell identification. Within the cells, black carbon particles were distinctly visible in small aggregates, enabling quantitative analysis of carbon particles.

Representative images of sputum macrophages are presented in Figure 1, depicting varying numbers of carbon particles in different cells. For instance, Figure 1A shows no carbon particles, while Figure 1D exhibits the highest concentration. The values of CCAM were measured as 0.00%, 2.24%, 6.64%, and 34.61% for macrophages (A-D), respectively. During the CCAM parameter determination process, a quantitative assessment of carbon content in each macrophage was conducted to mitigate biases arising from variations in macrophage size and the overlap of carbon particles with cell nuclei. CCAM calculation and analysis were performed for 50 macrophages per study subject, as shown in Figure 2.

The study selected carbon black packers (CBP) from a specific acetylene carbon black factory as the exposed group and recruited water pump workers from local water plants, who had no apparent occupational exposure to harmful factors, as the control group (Non-CBP). The results presented in Figure 2 indicate a higher CCAM in carbon black packaging workers compared to the control group.

Figure 1: Optical microscopic image showing carbon particles within airway macrophages. The black color in the cytoplasm represents carbon particles. Panels (A-D) depict representative images of airway macrophages, each exhibiting varying levels of CCAM. The CCAM values for macrophages in (A-D) were 0.00%, 2.24%, 6.64%, and 34.61%, respectively. The highest CCAM level was observed in (D). Scale bars: 10 µm. Please click here to view a larger version of this figure.

Figure 2: Quantitative levels of CCAM in the population. Fifty cytoplasmically intact and well-stained macrophages were randomly selected for analysis and quantification per study subject. Data were analyzed using Student's t-test: *P < 0.05. CBP: carbon black packers; Non-CBP: control group. Please click here to view a larger version of this figure.

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 procedures.

The determination of the carbon content of lung macrophages involves several steps, including the collection of lung macrophages, smearing, staining, photographing, and image analysis. Currently, there are two methods for collecting lung macrophages: induced sputum and lung lavage, each with its own advantages and limitations^18,19. Lung lavage can yield many homogeneous lung macrophages, but it is a traumatic sampling method that requires anesthesia and other medical conditions. Therefore, it is not suitable for large-scale general population sampling. In contrast, induced sputum is easy to perform, non-invasive, and suitable for large-scale sampling of the general population. It also allows for repeat samples to be obtained. Lung lavage obtains macrophages from alveoli or distal bronchioles, while induced sputum obtains macrophages that may originate from the airways. In this study, we performed induced sputum following the standard protocol published by the European Respiratory Society²⁰.

The success rate of induced sputum is influenced by various factors, including the concentration of nebulized inhaled saline and the presence of respiratory diseases in the patient^21,22,23. Sputum samples may also contain saliva, which can interfere with the measurement of macrophage carbon content, potentially reducing the count of airway macrophages in the sample²⁴. The operational procedures in this study were standardized, the methods were straightforward, and the reproducibility was high. It was observed that factors such as the size of the nebulized inhalation flow rate, the adequacy of induction time, and the presence of intervals during induction affected the success rate of induced sputum. These considerations are important for ensuring accurate and reliable measurement of CCAM as a biomarker for particulate matter exposure.

The Saccomanno fixative solution, consisting of 50% ethanol and 2% polyethylene glycol, is typically used as a fixative for preserving biological samples like bronchoalveolar lavage fluid. In this study, for the first time, this fixative solution was utilized to preserve sputum obtained from epidemiological field investigations. The results met the subsequent quantification of carbon in macrophages, akin to preserving sputum cells in the morning to obtain clear cancer cell smears²⁵. The operational procedures were standardized, the methods were straightforward, and the reproducibility was high. The advantages of induced sputum preservation in this study include (1) Easy configuration of fixative, (2) no refrigeration is needed for preservation, which is convenient for large-scale epidemiological surveys, and (3) samples can be preserved in a cool place away from light. Clear view under the microscope after staining, with identifiable nuclei, minimal impurities, and complete cell morphology. This is superior to direct sputum processing and smear application in terms of morphology²⁶.

Regarding experimental methods, the following details were noted: Sputum was periodically shaken during digestion in a 37 °C water bath until completely liquefied. After liquefaction, filtration on a 70 µm membrane was done before centrifugation to remove impurities for clearer staining background. Cellular precipitates were washed with DPBS again before smearing on slides. Excess liquid was removed during staining by washing slides with running water. Slide holders were changed between stain I and stain II to prevent residue. For photography, macrophages with complete morphology and good staining were randomly selected, affecting CCAM quantification²⁷.

Quantitative analysis software options for this study include Image SXM and Image J²⁸. Image SXM requires manual image editing before analysis to enhance accuracy, which significantly extends the required time. It is also limited to processing a single cell type. In contrast, the Image J software used in this study offers better specificity in image processing and broader compatibility. In other disciplines, high-content screening microscopy methods have been employed for the selection and subsequent analysis of fluorescently labeled cells, achieving automated high-throughput operations. The future direction of methodological research may involve developing automated labeling of macrophages in sputum and screening procedures²⁹. During Image J processing, factors influencing CCAM measurement include the measurer's knowledge of sample information and the determination of grayscale value thresholds. Adopting a blind approach can reduce observer bias, and some samples may need to be repeatedly measured to assess reproducibility. Independent image capture and analysis by two individuals with high-level expertise can enhance the accuracy of macrophage identification.

The study employed standard methods from the National Institute for Occupational Safety and Health (NIOSH) to assess the subjects' exposure to occupational particulate matter. Multiple evaluations were conducted on fine particulate matter (PM2.5) levels and PM2.5-related elemental carbon (EC) content in the carbon black packaging area and the work areas of the control group water plant workers. Up to 99.6% of carbon black (CB) particles had an aerodynamic diameter smaller than 2.5 µm, and 96.7% were smaller than 1.0 µm. In the autumn of 2012, the average PM2.5 level in the CB packing facility was 800 micrograms per cubic meter, with a PM2.5-related EC level of 657.0 micrograms per cubic meter^30,31. In the autumn of 2018, the geometric mean of CB was 637.4 µg/m³, and the PM2.5-related EC level was 364.6 µg/m³. In both observations, the EC/TC ratio exceeded 92.5%, consistent with the fact that CB is almost pure EC. Thus, it can be inferred from this study that carbon particles in macrophages are primarily derived from exposure to carbon black^30,31. As a result, the PM2.5 concentration in the working environment of the population exposed to carbon black in this study was 637.4 µg/m³, significantly higher than the PM2.5 concentration in the working environment of the control population (130.0 µg/m³) and about five times higher than that of the control population. Elemental carbon levels in the population exposed to carbon black were 364.6 µg/m³, much higher than the 2.0 µg/m³ in the control population.

In summary, this study utilized hypertonic saline-induced sputum to collect samples, obtained lung macrophages, made cell smears, and quantified the carbon content within 50 macrophages, using the median number to represent the level of macrophage carbon content for each individual. This research methodology can be adapted for large-scale population samples and used to accurately evaluate an individual's level of internal exposure to particulate matter.

Materials

Name	Company	Catalog Number	Comments
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