Final.html

Carbon Particles in Airway Macrophage as a Surrogate Marker in the Early Detection of Lung Diseases

 

NK Kalappanavar, CS VinodKumar, C Gouli, D Sanjay, K Nagendra, KG Basavarajappa, R Patil

Abstract

Background: It has been shown that inhalation of carbonaceous particulate matter may impair lung function in children.

Objective: Using the carbon content of airway macrophages as a marker of individual exposure to particulate matter derived from fossil fuel, we sought direct evidence for this association.

Methods: 300 children from puffed rice industrial areas and 300 children from population living in green zone were selected randomly. Airway macrophages were obtained from healthy children through sputum induction, and the grading of ultrafine carbon particles in airway macrophages was measured. Pulmonary function was also measured by spirometry.

Results: Pulmonary function tests showed that in industrial area 42.6% and 20.3% of children had moderate obstructive airway disease and restrictive airway disease, respectively. In the green zone area, 7% of children had obstructive airway disease and 6% had restrictive airway disease. Evaluation of airway macrophages for ultrafine carbon particles revealed that in industrial area there were ultrafine carbon particles of grade 2 in 23% of subjects and grade 3 in 8.33% of individuals with obstructive airway disease. In the green zone area, the rates were 1.67% and 0.7%, respectively.

Conclusion: The study provides a first evidence of the strong association between air pollution and development of airway diseases. Carbon particles in the sputum can be used as a marker for air pollution.

Keywords: Macrophages, alveolar; Particulate matter; Air pollutants; Spirometry; Lung diseases

Introduction

The incidence of allergic respiratory diseases and bronchial asthma appears to be increasing worldwide.1 People living in urban areas more frequently experience these conditions than those living in rural areas.2 One of the several causes of the rise in morbidity associated with allergic respiratory diseases is the increased presence of outdoor air pollutants resulting from more intense energy consumption and exhaust emissions from cars, other vehicles and industries.3,4 One of the important health problems is urban air pollution. It has been shown that air pollution can adversely affect lung function in patients with asthma.5 Air pollution can injure the airway mucous membranes and impair mucociliary clearance which in turn may facilitate access of inhaled allergens to the cells of the immune system, thus promoting sensitization of the airway.6 Consequently, a more severe immunoglobulin (IgE) mediated response to aeroallergens and airway inflammation could account for increasing prevalence of allergic respiratory diseases in polluted urban areas.7 Although air pollutants include a heterogeneous mixture of gases and particles, recent research studies have concerned the adverse effects of particulate matter (PM) which consists of primary particles, such as diesel soot or other combustion-derived particles emitted directly into the atmosphere, and secondary particles which are produced in the atmosphere through complex physicochemical reactions of various gases.8,9 Air-borne PM is generally defined based on the size distribution of the particles. Thus, PM10 and PM2.5 stand for PM with median aerodynamic diameters of less than 10 µm and 2.5 µm, respectively. Ultrafine particles as defined those particles with a diameter of <100 nm, are likely to deposit in human alveoli at a high rate.9 Inhaled air pollutants can cause lung inflammation, which can in turn alter the autonomic nervous control of heart rhythm and release of inflammatory mediators into the blood which affect extrapulmonary organs.10 Both pulmonary and systemic inflammation may occur through oxidative stress responses to reactive oxygen species. Another possibility is that ultrafine particles are able to translocate into the systemic circulation, and affect cardiovascular endpoints more directly.11

Among various states in India, Karnataka has some of the largest number of clusters of puffed rice units. It is estimated that there are about 2000 such units in Karnataka state and some of the major clusters are in the towns of Davangere, Hubli, Dharwad and Belgaum. Fuels used in the ovens for making puffed rice are mainly rice husk, wood, wood shavings, used automobile tire, groundnut shell, and agricultural residues. The used automobile tire is used in view of high heat it generates at low cost. Burning these fuels in highly inefficient conventional ovens generates high levels of particulate matter, carbon monoxide and other pollutants. We therefore conducted the present study to determine the role of carbon particles in airway macrophage and the pulmonary functions in children living in an industrial area and a green zone and to evaluate the utility of carbon particle in airway macrophage for early detection of respiratory disorders.

 

Materials and Methods

This study was conducted between September and December 2010. Ethical clearance was obtained from the Institutional Review Board.

Subjects

Inclusion criteria: Three-hundred apparently healthy children were randomly selected from a school with 380 students located in an industrial area. Another 300 apparently healthy children who served as comparison group were randomly selected from a school with 400 students located in a green zone far away from city, vehicular traffic and industrial area. Children were included if they were 8 to 16 years of age, were living in Davangere, and were living in the same house they had lived in for at least one year before the study began and if their parents reported that they had normal levels of activity and spirometry reading peak expiratory flow rate (PEFR)>80%.

Exclusion criteria: We excluded children who had any chronic respiratory condition, symptoms consistent with a respiratory infection over the previous three months, and those who were smokers. To ensure that the carbon content of airway macrophages reflected exposures in Davangere, we also excluded children who had spent more than five days outside the city in the previous three months as the half-life of particles in airway macrophages after a single instilled dose is 3.9 months.12,13

On the study day, children and parents were asked to complete a questionnaire that requested the child’s age, sex, number of siblings, race, and home address. The child’s body mass index (BMI) was calculated from the height and weight on that day.

Lung Function

Lung function was recorded no more than 20 minutes before sputum induction with the use of a spirometer (RMS Helios 401) and software. Spirometry was conducted according to the recommendations of the American Thoracic Society b a single operator.14 Forced vital capacity (FVC), forced expiratory flow between 25% and 75% of the FVC (FEF25-75) and the FEV1/FVC ratio were used as the primary measures to assess lung function. Each plot of the flow rate against volume during an FVC maneuver (flow volume curve) was visually examined for each child and if the final expiratory phase was stopped due to a Valsalva maneuver or hesitation, only the first portion of the curve was considered and only the FEV1 was calculated. If FEV1/FVC is >88%, then the patient has restricted lung disease; if it is ≤69% of the predicted value, then the patient has obstructive lung disease.14

Sputum Collection

Children were pre-treated with 200 µg salbutamol nebulization for five minutes and 5% saline nebulization for 15 minutes. Then, they were asked to cough out at least 2 mL of sputum in a wide mouthed clean container.15

Carbon Content of Airway Macrophages

Airway macrophages were visualized by light microscopy. The area occupied by black material (carbon) in each macrophage was assessed as previously described by an investigator who was blinded to pulmonary function test results. Smears were prepared and stained with hematoxylin and eosin.16 The smear was analyzed and grading of macrophage was done as follows. Grade 0: no carbon pigments in macrophage; grade 1: few carbon pigments in cytoplasm; grade 2: abundant carbon particles in cytoplasm, but nucleo-cytoplasmic differentiation maintained; and grade 3: macrophage flooded with carbon particles and nucleo-cytoplasmic differentiation lost.

Calculation of Particulate Matters at Different Study Centres

The annual mean level of primary PM10, that is the component of PM emitted directly from local source of combustion was calculated at industrial area and at green zone area by using ambient air quality monitoring instrument (Merck) a week before the subjects were enrolled into the study.

Statistical Analysis

Normality of data distribution was tested by Levene’s test. Unpaired Student’s t test was used to compare demographic features and pulmonary function test parameters. The categorical variables were analyzed by χ2 test. A p<0.05 was considered statistically significant. The data were analyzed by SPSS® 15.0 trial version.

 

Results

The numbers of children in the four strata were comparable in both groups. The male/female ratio was 65/35 in industrial area group and 62/38 in green area group. The mean±SD age of participants was 13.5±2.0 years for industrial area and 13.6±1.8 years for green zone. The mean annual level of primary PM10 concentration in industrial area and green zone was 1403 and 315 µg/m3, respectively.

BMI of children in industrial zone (15.9±2.8 kg/m2) was significantly lower than that of green zone students (18.5±4.2 kg/m2) (Table 1). There was no association between the carbon content of airway macrophages and age, weight, height, BMI, and level of activity. There was also no significant difference in carbon content of airway macrophages between boys and girls. More than 85% of children in industrial area that aged 8–10 years had carbon particles in their alveolar macrophages; it followed by children aged 11–13 years. Forty-two percent of children aged 14–17 years residing in green zone had more carbon particles in their alveolar macrophages when compared to other age groups.

 

Table 1: Demographic features of the studied children

Parameter

Mean±SD

p* value

Industrial area

(n=300)

Green zone

(n=300)

Age

13.5±2.0

13.6±1.8

0.52

Height (cm)

143.9±10.9

146.7±13.6

0.01

Weight (kg)

33.4±8.8

39.9±10.9

<0.001

BMI (kg/m2)

15.9±2.8

18.5±4.2

<0.001

Duration of residence in the study area (yrs)

10.4±2.2

10.0±1.1

0.14

Duration of stay at home (hrs)

12.6±1.4

12.9±1.7

0.21

The mean number of annual episodes of respiratory infections in children in industrial area and green zone was 11.8 and 3.3, respectively. The mean±SD linear height of children was 143.9±10.9 and 146.7±13.6 cm, respectively (Table 1).

While 72% of children in industrial area were found positive for carbon particles, 27% of children in green zone were found positive. Grades 2 and 3 carbon particles were seen in alveolar macrophage of 32% and 12% of children in industrial area, respectively. The prevalence rates in green zone children were 6% and 2%, respectively (Table 2).

 

Table 2: Frequency distribution of grades of carbon contents in alveolar macrophages stratified by two study areas.

Area

n

Grade

0

1

2

3

Green

300

189 (63.0%)

57(19.0%)

18 (6.0%)

06 (2.0%)

Industrial

300

84 (28.0%)

84 (28.0%)

96 (32.0%)

36 (12.0%)

There was an inverse, dose-dependent association between the carbon content of airway macrophages and pulmonary function: each increase in the grade of carbon content was associated with a reduction of 17% in FEV1, 12.9% in FVC, and 34.7% in FEF25-75. There was no association between the carbon content of airway macrophages and the FEV1/FVC ratio (Tables 3, 4). These associations remained significant when lung function was measured 15 minutes after the administration of an inhaled bronchodilator.

 

Table 3: Measured pulmonary function test parameters recorded in study groups

Parameters

Industrial area

Green zone

p* value

Range

Median

Mean±SD

Range

Median

Mean+/-SD

FVC

60–116

78

83.4±19.2

66–120

84

84.3±12.7

0.56

FEV1

74–144

88

97.8±24.0

77–129

100

99.2±13.8

0.35

FEV1/FVC

108–125.0

115

117.1±5.4

108–125

116

117.8±4.7

0.52

FEF25-75

53–167

105

105.5±24.1

65–176

100

103.1±25.8

0.33

Table 4: Number (%) of children stratified by pulmonary function test results and living areas

Parameters

Severity

Industrial

Green zone

p value

FEV1

>80

172 (57.3)

279 (93.0)

<0.001

60–80

128 (42.6)

21 (7.0)

≤60

0 (0.0)

0 (0.0)

FEV1/FVC

≥88

61 (20.3)

18(6.0)

0.08

70–87

168 (56.0)

276 (92)

<69

71 (23.6)

6 (2.0)

FVC

>80

142(47.3)

219 (73.0)

<0.001

60–80

145(48.3)

81 (27.0)

45–65

13 (4.3)

0 (0.0)

<45

0 (0.0)

0 (0.0)

Based on pulmonary function test results, 42.6% of children in industrial area had moderate obstructive airway disease; the prevalence was 7% in green zone. Of children in industrial area, 20.3% had restrictive airway disease; the prevalence was 6% in the green zone (p<0.05).

Of those children in industrial area who had restrictive airway disease, 22 had grade 2, and 10 had grade 3 carbon particles in airway macrophages. In green zone children only one had grade 2 carbon particles (Table 5).

 

Table 5: Number of children with various grades of alveolar macrophage carbon contents stratified by FEV1/FVC and study area

FEV1/FVC

Industrial area

Green zone

Grade

Grade

1

2

3

1

2

3

≥88

0

22

10

0

3

0

70–87

80

5

1

51

0

0

<70

4

69

25

2

5

2

p<0.001

p=0.01

In industrial group, airway macrophages carbon particles of grade 2 and grade 3 were observed in 69, and 25, children, respectively—all of them had obstructive airway disease. In green zone group, airway macrophages carbon particles of grade 2 were observed in five and three children who also had obstructive airway disease.

 

Discussion

In developed countries, modernization has been accompanied by a shift from biomass fuels such as wood to petroleum products and electricity. In developing countries, however, even where cleaner and more sophisticated fuels are available, the small scale industries often continue to use simple biomass fuels. Although the proportion of global energy derived from biomass fuels fell from 50% in 1900 to around 13% in 2000, there is evidence that their use is now increasing among the poor.17 Poverty is one of the main barriers to the adoption of cleaner fuels. The slow pace of development in India suggests that biomass fuels will continue to be used by the poor for many decades.

Biomass fuel is any material derived from plants or animals which is deliberately burnt by humans. Wood is the most common example, but the use of animal dung and crop residues is also widespread. China, South Africa and some other countries also use coal extensively for domestic needs. Used vehicle tires are burnt which may cause allergy either by direct contact with latex products or inhalation of air-borne allergens; such allergies are frequently termed “latex allergy.” Latex is primarily recognized as an indoor allergen. A major component of lorry tires is natural rubber latex. Tire dust in urban air samples has been postulated to contribute to latex sensitization and asthma.18

In the present study the mean duration of exposure to outdoor pollutants was similar in both study groups. Earlier studies showed that the predominant source of carbon particles in alveolar macrophages is from combustion of fossil fuels. In our study, we found that the likelihood of detecting carbon particles in alveolar macrophages as well as its grade is higher in children residing in industrial area than green zone.

We also found a statistically significant difference in the mean number of annual respiratory episodes in children in industrial area than in green zone. Similar observations were made in earlier studies conducted in India. However, in contrast to earlier reports, we did not observe any statistically significant difference in PEFR.19

Based on pulmonary function test results, 42.6% of children living in industrial area compared to 7% of those in green zone had moderate obstructive airway disease. Twenty percent of children in industrial area compared to 6% in children in green zone had restrictive airway disease. The number of children in industrial area who had grades 2 or 3 alveolar carbon particles, was significantly higher than those in green zone.

We could not figure out whether the inverse association observed between the grade of carbon content in airway macrophages and lung function parameters represents a short-term or a long-term effect on lung function. Our results have, however, ruled out short-term reversible bronchoconstriction; significant inverse associations remained after bronchodilator therapy.

It is also unclear whether the carbon content of airway macrophages reflects long-term or short-term exposure to PM. Some carbon particles could have been acquired several months before our analysis, since soot has been observed in airway macrophages even 10 months after a brief occupational exposure, and insoluble particles would remain in airway macrophages for up to three months after instillation in experimental studies. Furthermore, the carbon content of airway macrophages may not reflect the content in more distal alveolar cells, since sputum induction samples macrophages from the larger airways.

It has been shown in experimental exposure studies that increased level of activity, lower age, and higher BMI are associated with increased deposition of particles in the lower airway. In the present study, none of these variables were associated with carbon contents of airway macrophages. However, we found that carbon contents in the airway macrophages of children living in an industrial area where primary PM10 were abundant was higher than those living in a green zone.

Increased vulnerability to bacterial infection of the lower respiratory tract is a hallmark of COPD.19 Recent data from human bronchial epithelial cells exposed to cigarette smoke suggest that this may, in part, be due to suppression of antibacterial host defense.20 Similarly, in children, there is good evidence that exposure to PM increases vulnerability to bacterial infection.9 This association between PM and bacterial infection in children is important because exposure to PM is ubiquitous and infection is common, with 156 million new episodes of pneumonia per year in young children worldwide (151 million of these are in the developing countries);21 10% of these episodes are life-threatening.21 A recent meta-analysis of studies performed in the developing world estimated that the odds ratio for severe pneumonia in children under five years of age exposed to smoke from biomass and other high PM-emitting fuels is 1.78.22 Few studies in the developed countries have assessed the association between PM and vulnerability of children to bacterial infection. Even fewer studies of environmental exposures and infection have identified casual pathogens. However, Streptococcus pneumoniae is likely to be important, since it is the most common etiologic agent for pneumonia in young children.23,24

To conclude, the presence and the amount of carbon particles in airway macrophages can be used as an early marker of altered pulmonary functions. Also the higher the load of carbon particles in airway macrophages, the more severe alteration in pulmonary function test parameters. In this study, those with carbon particles in their alveolar macrophage have shown signs of both obstructive airway disease and restrictive airway disease. It reflects that there is a need for further research to shed light over the correlation between pollution and lung development both in utero and post-natal period.

 

Conflicts of Interest: None declared.

 

References

  1. Host A, Halken S. The role of allergy in childhood asthma. Allergy 2000;55:600-8.
  2. Marks GB, Tovey ER, Green W, et al. The effect of changes in house dust mite allergen exposure on the severity of asthma. Clin Exp Allergy 1995;25:114-8.
  3. Atkinson RW, Anderson HR, Sunyer J, et al. Acute effects of particulate air pollution and health; a European approach. Am J Resp Crit Med 2001;164:1860-6.
  4. Walter S, Miles J, Ayres J, Archer G. Effect of an air pollution episode on respiratory functions on patients wit asthma. Thorax 1993;48:1063-8.
  5. Trindade JC. The importance of diagnosis of allergy in early wheezing. Pediatr Allergy Immunol 1998;9:23-29.
  6. Sasai K, Furukawa S, Muto T, et al. Early detection of specific IgE antibody against house dust mite in children at risk of allergic disease. J Pediatr 1996;128:834-40.
  7. Durham SR, Craddock CF, Cookson WO, Benson MK. Increases in airway responsiveness to histamine precede allergen-induced late asthmatic responses. J Allergy Clin Imunol 1988;82:764-70.
  8. Stone V. Environmental air pollution. Am J Respir Crit Care Med 2000;162:44-7.
  9. Pierse N, Rushton L, Harris RS, et al. Locally generated particulate pollution and respiratory symptoms in young children. Thorax 2006;61:216-20.
  10. Olivieri D, Pesci A, Bertorelli G. Immunological defences in airways: from injury to repair. Lung Biology in Health and Disease. New York, Marcel Dekker inc 1996;93:43-69.
  11. Blomberg A, Krishna MT, Bocchino V, et al. The inflammatory effects of 2 ppm NO2 on the airways of healthy subjects. Am J Respir Crit Care Med 1997;156:418-24.
  12. Fireman EM, Lerman Y, Ganor E, et al. Induced sputum assessment in New York City firefighters exposed to World Trade Center dust. Environ Health Perspect 2004;112:1564-9.
  13. Lay JC, Bennett WD, Kim CS, et al. Retention and intracellulardistribution of instilled iron oxide particles in human alveolar macrophages. AmJ Respir Cell Mol Biol 1998;18:687-95.
  14. American Thoracic Society. Standardization of spirometry, 1994 update. Am J Respir Crit Care Med 1995;152:1107-36.
  15. Cataldo D, Foidart JM, Lau L, et al. Induced sputum: comparison between isotonic and hypertonic saline solution inhalation in patients with asthma. Chest 2001;120:1815-21.
  16. Bancroft JD, Gamble M. Theory and practice of histological techniques. 5th Ed. Philadelphia: Churchill Livingstone, 2002.
  17. Burr ML, Butland BK, King S, et al. Changes in asthma prevalence: two surveys 15 years apart. Arch Dis Child 1989;64:1452-56.
  18. Devereux G, Ayatollahi T, Ward R, et al. Asthma, airway responsiveness and air pollution in two contrasting districts of Northern England. Thorax 1996;51:I69-74.
  19. Peters A, Dockery DW, Heinrich J, Wichmann HE. Short-term effects of particulate air pollution on respiratory morbidity in asthmatic children. Eur Respir J 1997;10:872-9.
  20. Ostro BD, Lipsett MJ, Mann JK, et al. Air pollution and respiratory morbidity among adults in southern California. Am J Epidemiol 1993;137:691-700.
  21. Delfino RJ, Zeiger RS, Seltzer JM, et al. Association of asthma symptoms with peak particulate air pollution and effect modification by anti-inflammatory medication use. Environ Health Perspect 2002;110:607-17.
  22. Pilotto LS, Douglas RM, Attewell RG, Wilson SR. Respiratory effects associated with indoor nitrogen dioxide exposure in children. Int J Epidemiol 1997;26:788-96
  23. Fusco D, Forastiere F, Michelozzi P, et al. Air pollution and hospital admissions for respiratory conditions in Rome, Italy. Eur Respir J 2001;17:1143-50
  24. Anderson HR, Spix C, Medina S, et al. Air pollution and daily admissions for chronic obstructive pulmonary disease in 6 European cities: results from the APHEA project. Eur Respir J 1997;10:1064-71

TAKE-HOME MESSAGE

  • The incidence of allergic respiratory diseases and bronchial asthma are increasing worldwide.
  • The predominant source of carbon particles in alveolar macrophages is from combustion of fossil fuels.
  • Carbon particles in alveolar macrophages as well as its grade is higher in children residing in industrial area than green zone.

Cite this article as: Kalappanavar NK, VinodKumar CS, Gouli C, et al. Carbon particles in airway macrophage as a surrogate marker in the early detection of lung diseases. The International Journal of Occupational and Environmental Medicine 2012;3:68-75.




 pISSN: 2008-6520
 eISSN: 2008-6814

Creative Commons LicenseThis work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License