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Inflammation
ISSN 0360-3997
Volume 35
Number 2
Inflammation (2012) 35:671-683
DOI 10.1007/s10753-011-9360-2
Neutrophilic Inflammatory Response
and Oxidative Stress in Premenopausal
Women Chronically Exposed to Indoor Air
Pollution from Biomass Burning
Anirban Banerjee, Nandan Kumar
Mondal, Debangshu Das & Manas
Ranjan Ray
1 23
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Neutrophilic Inflammatory Response and Oxidative Stress
in Premenopausal Women Chronically Exposed to Indoor
Air Pollution from Biomass Burning
Anirban Banerjee,
1
Nandan Kumar Mondal,
1,2
Debangshu Das,
1
and Manas Ranjan Ray
1
Abstact—The possibility of inflammation and neutrophil activation in response to indoor air poll-
ution (IAP) from biomass fuel use has been investigated. For this, 142 premenopausal, never-
smoking women (median age, 34 years) who cook exclusively with biomass (wood, dung, crop
wastes) and 126 age-matched control women who cook with cleaner fuel liquefied petroleum gas
(LPG) were enrolled. The neutrophil count in blood and sputum was significantly higher (p< 0.05)
in biomass users than the control group. Flow cytometric analysis revealed marked increase in the
surface expression of CD35 (complement receptor-1), CD16 (F
C
γreceptor III), and β
2
Mac-1
integrin (CD11b/CD18) on circulating neutrophils of biomass users. Besides, enzyme-linked imm-
unosorbent assay showed that they had 72%, 67%, and 54% higher plasma levels of the proinfla-
mmatory cytokines tumor necrosis factor-alpha, interleukin-6, and interleukin-12, respectively, and
doubled neutrophil chemoattractant interleukin-8. Immunocytochemical study revealed significantly
higher percentage of airway neutrophils expressing inducible nitric oxide synthase, while the serum
level of nitric oxide was doubled in women who cooked with biomass. Spectrophotometric analysis
documented higher myeloperoxidase activity in circulating neutrophils of biomass users, suggesting
neutrophil activation. Flow cytometry showed excess generation of reactive oxygen species (ROS)
by leukocytes of biomass-using women, whereas their erythrocytes contained a depleted level of
antioxidant enzyme superoxide dismutase (SOD). Indoor air of biomass-using households had two
to four times more particulate matter with diameters of <10 μm (PM
10
) and <2.5 μm (PM
2.5
)as
measured by real-time laser photometer. After controlling potential confounders, rise in proin-
flammatory mediators among biomass users were positively associated with PM
10
and PM
2.5
in
indoor air, suggesting a close relationship between IAP and neutrophil activation. Besides, the levels
of neutrophil activation and inflammation markers were positively associated with generation of
ROS and negatively with SOD, indicating a role of oxidative stress in mediating neutrophilic inf-
lammatory response following chronic inhalation of biomass smoke.
KEY WORDS: biomass fuel; neutrophil; inflammation; oxidative stress; premenopausal women; India.
INTRODUCTION
A large majority of rural women of the developing
world including India are chronically exposed to high
levels of indoor air pollution (IAP) from daily household
cooking with traditional biomass fuel (BMF) such as
dried cow dung cake, firewood, and agricultural wastes.
Smoke emitted from burning biomass contains a wide
ABBREVIATIONS: ACD, acid citrate dextrose; BMF, biomass fuel;
BSA, bovine serum albumin; DCF-DA, dichlorofluorescein diacetate;
EDTA, ethylenediaminetetraacetic acid; ELISA, enzyme-linked immu-
nosorbent assay; FACS, fluorescence-activated cell sorter; FITC,
fluorescein isothiocyanate; HRP, horseradish peroxidase; IAP, indoor
air pollution; ICC, immunocytochemistry; IL, interleukin; iNOS,
inducible nitric oxide synthase; LPG, liquefied petroleum gas; MFI,
mean fluorescence intensity; MPO, myeloperoxidase; NO, nitric oxide;
PAP, Papanicolaou; PBS, phosphate-buffered saline; PE, phycoerythrin;
PM, particulate matter; ROS, reactive oxygen species; SOD, super-
oxide dismutase; TNF, tumor necrosis factor
Anirban Banerjee and Nandan Kumar Mondal contributed equally to
this work.
1
Department of Experimental Hematology, Chittaranjan National
Cancer Institute, 37, S. P. Mukherjee Road, Kolkata, 700 026, India
2
To whom correspondence should be addressed at Department of
Experimental Hematology, Chittaranjan National Cancer Institute, 37,
S. P. Mukherjee Road, Kolkata, 700 026, India. E-mail: nandan_
gm@yahoo.com
0360-3997/12/0200-0671/0 #2011 Springer Science+Business Media, LLC
Inflammation, Vol. 35, No. 2, April 2012 (#2011)
DOI: 10.1007/s10753-011-9360-2
671
Author's personal copy
spectrum of pollutants that include carbon monoxide,
coarse, fine, and ultrafine particulate matters (PM),
oxides of nitrogen and sulfur, formaldehyde, transitional
metals, and volatile organic compounds including
benzene and polycyclic aromatic hydrocarbons such as
benzo(a)pyrene [1]. Some of these pollutants like benzo
(a)pyrene and benzene are potent human carcinogens
[2]. Women who used to cook with BMF in poorly
ventilated kitchen for 2–6 h/day are believed to be
inhaling carcinogens equivalent to smoking two packs of
cigarettes per day [3]. Epidemiological studies have
shown that exposure to fine particles promotes inflam-
mation in the lung [4–9] via activation of alveolar
macrophages and lung epithelial cells [10,11]. Such
exposures may also elicit systemic inflammatory
response [12], associated with increased levels of several
cytokines (interleukin-6 and -1b) in the bloodstream [13]
as well as increased production, release, and activation
of neutrophils from the bone marrow [14].
Neutrophils are the most abundant leukocytes
present in human blood. These cells are important for
phagocytosis and destruction of invading pathogens,
especially bacteria [15]. They are among the first cells to
leave the bloodstream and reach the tissues in case of
tissue injury [16]. During the beginning of an acute
phase of inflammation, particularly as a result of
bacterial infection, environmental exposure, and some
cancers, neutrophils are among the first responders that
migrate towards the site of inflammation. Such migration
of blood leukocytes to the tissues in order to fight
against invading pathogens and to mediate tissue repair
is known as inflammation. Neutrophils have been
implicated in the development of bronchial hyperres-
ponsiveness [17]. Neutrophilic inflammation in the
airways and the bronchial submucosa has been reported
in patients with severe asthma [18,19], especially during
exacerbation [20]. Neutrophil elastase has the capacity to
induce mucus gland hyperplasia and mucus secretion
[21] and proliferation or apoptosis of airway smooth
muscle cells [22,23]. Myeloperoxidase (MPO) is an
enzyme found in myeloid cells, particularly in neutro-
phils, and to a lesser extent in monocytes and tissue
macrophages. It plays an important role in the host
defense against bacteria and viruses. In neutrophils,
MPO is located within the primary granules and its
concentration is used as an indicator of neutrophil
activation and corresponding inflammatory response
[24]. Neutrophil migration from circulation into an area
of inflammation involves regulated expression of a
number of adhesion molecules on the cell surface.
Upregulation of these surface molecules has been
reported in patients with chronic obstructive pulmonary
disease (COPD) [25]. A recent study has shown allergic
pulmonary inflammation following exposure to diesel
exhaust particles [26]. Moreover, studies in laboratory
animals have documented lung inflammation induced by
air pollution [27]. In view of these reports, it is
reasonable to assume that IAP from biomass burning
may lead to the activation of circulating neutrophils and
consequent inflammatory response in women who cook
with these fuels.
Neutrophils in peripheral blood may be activated or
primed with regard to superoxide production, chemotaxis,
and increased expression of surface molecules such as
CD35 which mediates the binding and phagocytosis of
C3b-coated particles and immune complexes [28]. The
study of Smith et al.[29] suggested that upregulation of
CD11b expression may be associated with neutrophils
degranulation. Data from studies of pathological condi-
tions suggest that neutrophil receptor expression is altered
in response to inflammatory stimuli and tissue injury. For
example, the expression of CD11b and CD35 receptors is
enhanced in patients suffering sepsis. In contrast, the
expression of CD11b, CD35, and CD16 is reduced in
patients who have experienced acute trauma and burns.
These alterations may represent adaptive responses to
control the level of neutrophils and complement activation
during these pathological conditions [30–32]. Alterations
in neutrophil receptor expression could also affect neu-
trophil functional activity. For example, a reduction in the
neutrophil expression of CD11b and CD16 in patients with
thermal injuries has been associated with defects in
respiratory burst activity, and this has resulted in an
increased susceptibility to infection [33]. Neutrophils
respond to infection and tissue injury by recognizing and
binding immunoglobulin G (IgG) molecules and comple-
ment proteins that coat the surface of foreign pathogens
and host tissue fragments. This recognition-binding proc-
ess is mediated by neutrophil surface receptors, including
the complement receptor 1 CD35, the complement
receptor 3 CD11b, and the low-affinity IgG receptor
CD16. The engagement of these surface receptors initiates
a cascade of intracellular events leading to the release of
enzymes (degranulation) and reactive oxygen species
(ROS; respiratory burst activity) from neutrophils [34].
Together, these enzymes and ROS assist in the destruction
and degradation of foreign pathogens and damaged tissue
fragments [35].
Our earlier studies have linked IAP from BMF with
upregulation of ribosome biogenesis [36,37], inappro-
672 Banerjee, Mondal, Das, and Ray
Author's personal copy
priate response of DNA damage repair proteins in the
face of increased DNA damage [38,39], overactivation
of signal transduction pathway involving Akt, i.e.,
protein kinase B [40], and hypertension with elevated
levels of oxidized low-density lipoprotein indicating
cardiovascular disease [41]. These findings along with
observations from other studies led us to hypothesize
that chronic exposure to IAP from biomass burning
could be associated with neutrophilic inflammatory
responses and oxidative stress in premenopausal women
of rural India who cook regularly with BMF. To our
knowledge, no such study has been carried out in India
or any other developing country where biomass is
extensively used as a major source of household energy.
Against this background, we examined in this study the
impact of IAP from BMF use on neutrophil activation in
a group of never-smoking, premenopausal women from
eastern India who used to cook exclusively with BMF
for the past 5 years or more. We have compared the
findings with an age-matched group of control women
from same neighborhood who cooked with cleaner fuel
liquefied petroleum gas (LPG).
MATERIALS AND METHODS
Subjects
A total of 268 premenopausal women aged between
23 and 41 years from rural areas of West Bengal, a state
in eastern India, were enrolled in this study after
obtaining written informed consent. They attended
health checkup camps organized in different villages
with the active cooperation of the local administrative
bodies and nongovernmental organizations. Among the
participants, 142 women (age, 23–40 years; median age,
34 years) were cooking daily for 2.5–5.5 h exclusively
with wood, cow dung, and agricultural refuse, such as
bamboo, jute stick, paddy husk, hay, and dried leaves for
the past 5 years or more. Accordingly, they were
grouped as biomass users. The remaining 126 women
(age, 24–41 years; median age, 33 years) were from the
same villages but they used to cook with cleaner fuel
LPG and were considered as control.
Inclusion and Exclusion Criteria
The inclusion criteria were apparently healthy,
premenopausal married women actively engaged in
household cooking for the past 5 years or more, who
were nonsmokers and nonchewers of tobacco, and had a
body mass index (BMI) >15 and <30 kg/m
2
. Women
using oral contraceptive, had a history of malignancy, or
were currently under medication were excluded.
During personal interview with female members of
the research team, each participant was requested to
furnish information about age, education, family size
and income, habit, cooking time per day, years of
cooking, fuel and oven type, ventilation and location of
kitchen, and general health problems in the past 3 months
and last 1 year. As most of the participants were poorly
educated, the researchers recorded their responses in
structured questionnaire forms on their behalf. The
Ethics Committee of Chittaranjan National Cancer
Institute approved the study protocol.
Collection of Blood and Expectorated Sputum
Venous blood (5 ml) was collected in vacutainer tubes
(Becton Dickinson [BD], USA) containing K
2
EDTA as
anticoagulant. Blood was collected at a fixed time of the
day (09:30 AM –10:30 AM) to minimize diurnal variation.
A drop of blood was used for smear preparation on clean
glass slide for each individual. An aliquot of whole blood
was mixed with acid citrate dextrose (ACD) solution in the
ratio of 5:1 separately in sterile vacutainer tubes for
isolation of circulation neutrophils. Blood in the no-
additive vacutainer was allowed to clot and serum was
collected. Plasma was obtained by centrifugation at
2,500×gfor 10 min at 4°C.
Early morning expectorated sputa were collected in
sterile plastic cups for three consecutive days to harvest
airway cells following the procedure of Erkilic et al.
[42]. Two smears were prepared from the nontransparent
highly viscous part of each freshly collected sputum
sample on clean glass slides from each day’s sample—
one for Papanicolaou (PAP) staining and the other for
immunocytochemistry (ICC) of inducible nitric oxide
synthase (iNOS). After air drying, one slide marked for
PAP staining was fixed with 95% ethanol and the other
for ICC were fixed in ice-cold methanol for 20 min at
the site of collection. The remaining part of the
expectorated sputum was collected in sterile plastic
screw-cap tubes containing 20 ml of phosphate-buffered
saline (PBS) with 0.1% dithiotheritol.
Routine Hematology
Differential counts of leukocytes were done from
Leishman-stained blood films. Routine hematological
parameters, such as total and differential counts of white
blood cells and total count of platelets, were carried out
673Neutrophilic Inflammatory Response and Oxidative Stress
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using a hemocytometer under light microscope (Leitz,
Germany).
PAP Staining for Routine Sputum Cytopathology
Ethanol-fixed slides were brought to the laboratory
and were stained with PAP following the procedure of
Hughes and Dodds [43]. The slides were coded and
examined under light microscope (Leitz, Germany) at
×400 and ×1,000 magnifications. At least 10 high-power
fields (hpf; ×40 objective and ×10 eyepiece) per slide
were examined. Sputum cytology and differential dis-
tribution of inflammatory cells were performed follow-
ing the established criteria [44].
Isolation of Circulating Neutrophils
Neutrophils were isolated following the procedure
of Drabikova et al.[45] with slight modifications. In
brief, blood in ACD was added with 6% dextran
solution in 0.9% NaCl (blood–dextran ratio, 1:2 v/v).
Then, it was allowed to sediment for 45 min at room
temperature. The supernatant was centrifuged at 700×g
for 12 min at 4°C, the cell pellet was reconstituted in
1.2 ml ice-cold distilled water, and within 20 s, 400 μlof
0.6 M KCl was added and mixed. It was diluted to 5 ml
with PBS and kept in ice for 5 min for red blood cell
(RBC) lysis. It was then centrifuged at 770×g for 6 min
at 4°C. Supernatant was discarded and pellet resus-
pended in 1–2 ml of ice-cold PBS. Next, the cell
suspension was layered over 3 ml of Ficoll–Histopaque
(Sigma Aldrich, USA) in a 15-ml tube and centrifuged at
1,200×g for 30 min. After discarding the supernatant,
the pellet was dissolved in 500 μl of ice-cold Hank’s
balanced salt solution. About 99% of neutrophils were
isolated by this procedure and viability of the cells was
>90% in trypan blue dye exclusion test.
Assessment of the Expression of Surface Molecules
by Flow Cytometry
Whole blood (25 μl) was added to polypropylene
tubes and incubated for 20 min at room temperature in
the dark with FITC-conjugated antihuman CD11b,
CD18, and CD35 monoclonal antibodies, PE-conjugated
antihuman CD16 monoclonal antibody (BD, USA), and
isotype controls. Thereafter, the erythrocytes were lysed
with 2 ml of 1X FACS lysing solution (BD, USA), and
samples were centrifuged at 500×gfor 5 min. The cell
pellet was washed with ice-cold PBS containing 0.1%
sodium azide and resuspended in 500 μlof1%
paraformaldehyde in PBS. The samples were analyzed
in a flow cytometer (FACSCalibur with Sorter, BD, San
Jose, CA, USA) using Cell Quest software (BD, USA).
A gate was drawn around the granulocytes showing
neutrophils as CD16-positive cells with eosinophils as
CD16-negative cells with high side scatter. A further
gate was then drawn around the neutrophil (CD16+) or
eosinophil (CD16−) populations and reflected into a
histogram so that cells could be labeled with a further
fluorochrome (e.g., CD11b or CD18). Fluorescence was
measured with a FACSCalibur flow cytometer and Cell
Quest software (BD, USA). Measurements were made
on the FL1 (green) and FL2 (red) channels, and the gates
were adjusted to the negative control quadrant. A total of
15,000 events were collected. Results were recorded as
mean fluorescence intensity (MFI), which represents the
cell surface receptor density.
Measurement of Proinflammatory Cytokine
and Chemokine by ELISA
The levels of proinflammatory cytokine tumor
necrosis factor-alpha (TNF-α), interleukin-6 (IL-6),
interleukin-12 (IL-12), and chemokine interleukin-8
(IL-8) were measured in blood plasma and nitric oxide
(NO) level in serum by ELISA using commercially
available kits of BD Biosciences Pharmingen, San
Diego, USA (for TNF-α, IL-6, and IL-12), Roche
Diagnostics GmbH, Germany (for IL-8), and Oxford
Biomedical Research, Oxford, MI, USA (for NO). The
assays were performed following the procedures recom-
mended by the manufacturers. The instructions of the
manufacturers were followed during the study and
readings were taken in microplate reader (Bio-Rad,
USA) at 450 nm.
Immunocytochemical Localization of iNOS
ICC of iNOS in isolated blood neutrophils and
sputum cells were done following a standard procedure
[38,40]. In brief, slides were air dried and fixed in ice-
cold methanol for 30 min, air dried, washed in PBS
thrice, and blocked in 3% BSA (Sigma-Aldrich Chem-
icals, Saint Louis, MO, USA) for 1 h at room temper-
ature. The slides were overnight incubated in darkness
with rabbit polyclonal iNOS (Santa Cruz, CA, USA)
primary antibody (diluted 1:100 in 1% BSA, respec-
tively) in a humid box at 4°C. After washing with PBS,
slides were incubated with antirabbit IgG, F(ab′)2-HRP
(Santacruz, USA) secondary antibody diluted 1:500 in
1% BSA for 90 min. The slides were developed by
674 Banerjee, Mondal, Das, and Ray
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incubating with substrate for HRP for 45 min in
darkness followed by washing with distilled water and
counterstaining with hematoxylin, dehydration in graded
ethanol, and mounted in distrene plasticizer xylene. The
slides were coded and examined blindly. For each
participant, one blood neutrophil sample and three sputa
samples obtained from three consecutive days were
examined. The 3-day average values of iNOS expres-
sions in sputum cells were taken as the representative
data of each participant.
Quantitative Measurement of MPO Activity
by Spectrophotometry
MPO activity in isolated blood neutrophil homoge-
nate was determined by a modification of the assay
method involving O-dianisidine [46]. The principle
involves oxidation of O-dianisidine by MPO present in
the isolated neutrophil homogenate to produce a color
complex which is read spectrophotometrically at
460 nm. The higher the MPO activity, the more oxygen
released from hydrogen peroxide; hence, more oxidation
of O-dianisidine and consequently higher observed
spectrophotometer reading at 460 nm. The assay mixture
contained 0.3 ml of 0.1 M phosphate buffer (pH 6.0),
0.3 ml of 0.01 M H
2
O
2
, 0.5 ml 0.02 M of O-dianisidine
(Sigma Chem, USA; freshly prepared in deionized
water), and 10 μl neutrophil homogenate in a final
volume of 3 ml. The neutrophil homogenate was added
last in a cuvette with a path length of 1 cm and the
change in absorbance in 460 nm was followed for
10 min. One unit of MPO is defined as that giving an
increase in absorbance of 0.001 per minute and specific
activity is given as international units per milligram of
protein.
Flow Cytometric Measurement of ROS Generation
Generation of ROS was measured in leukocytes
present in anticoagulated venous blood and sputum cells
by flow cytometry using DCF-DA (Sigma Chem, USA)
following the procedure of Rothe and Valet [47] with
slight modifications [40]. For this, 10,000 events were
acquired in a flow cytometer (FACSCalibur with sorter,
Becton Dickinson [BD], San Jose, CA, USA) using Cell
Quest software (BD). Respiratory burst and generation
of ROS by cells were associated with emission of green
fluorescence that was recorded in fluorescence channel 1
and was expressed as MFI in arbitrary units. In case of
blood, granulocytes, monocytes, and lymphocytes were
gated on the basis of their characteristic forward and side
scatters on dot plot and the MFI of each population was
recorded, while the MFI of the total cells present in
sputum was recorded.
Spectrophotometric Measurement of SOD
The activity of the antioxidant enzyme superoxide
dismutase (SOD) was assayed in blood erythrocytes and
sputum cell lysate spectrophotometrically following the
procedure of Paoletti et al.[48]. Whole sputum collected
in PBS with 0.1% dithiotheritol were used for cell lysate
preparation for SOD assay followed by our previously
published procedure [40]. Lysates were centrifuged at
15,000×gfor 10 min at 4°C in an Eppendorf micro-
centrifuge. The supernatant was collected and used for
SOD assay. Ethylenediaminetetraacetic acid (EDTA)-
anticoagulated blood was centrifuged at 200×gat 4°C
for 5 min, the RBC pellet was collected and washed in
0.9% saline and then lysed with cold distilled water (1:9,
v/v). Following centrifugation at 500×gfor 10 min, the
supernatant was collected. In spectrophotometric cuv-
ette, 800 ml of triethanolamine (TEA)–diethanolamine
(DEA) buffer containing 100 mM each of TEA and
DEA (Qualigens, Mumbai, India), 40 ml of 7.5 mM
nicotinamide adenine dinucleotide reduced disodium salt
(NADH, SRL, Mumbai, India), pH 7.4, 25 ml of a
mixture (1:1, v/v) of 0.2 M EDTA disodium salt (Sigma-
Aldrich Chem, St. Louis, MO, USA) and 0.1 M
manganous chloride (SD-Fine Chem, Mumbai, India),
and 100 ml of sample (RBC or sputum cell lysate) were
added and mixed well. The absorbance (OD) was
measured at 340 nm in a spectrophotometer (Shimadzu,
Kyoto, Japan) immediately (0 min) and 1, 2, 3, 4, and
5 min after the addition of mercaptoethanol. SOD
activity was calculated from the standard curve and
was expressed as units per milliliter.
Measurement of PM
10
and PM
2.5
in Indoor Air
PM with aerodynamic diameter less than 10 mm
(PM
10
) and 2.5 mm (PM
2.5
) were measured in the
cooking areas with real-time laser photometer (Dust-
Trak™Aerosol Monitor, model 8520, TSI Inc., Shore-
view MN, USA) that contained 10-mm nylon Dor-
Oliver cyclone and operated at a flow rate of 1.7 L/min,
measuring particle load in the concentration range of 1
to 100 mg/m
3
. The monitor was calibrated to the
standard ISO 12103-1 A1 test dust. We used two
monitors for simultaneous measurement of PM
10
and
PM
2.5
. Air sampling was done in each household for
three consecutive days, 8 h/day (07:00 AM –03:00 PM)
675Neutrophilic Inflammatory Response and Oxidative Stress
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covering both cooking and noncooking time. The mean
of 3 days was used as the indoor air quality of a single
household. Biomass-using women cook in a sitting
position 2–3 ft away from the open chullah (oven).
Accordingly, the monitor was placed in the breathing
zone of the cook 2.5 ft above the floor level on a wooden
stool 3 ft away from the chullah. LPG users, on the other
hand, cook in a standing position, and the monitor was,
therefore, placed at a height of 4.5 ft. Since laser
photometers make an overestimation of PM levels by
twofold to threefold [49], the raw data require reduction
using correction factors. We reduced the 8-h raw data by
dividing with a correction factor of 2.5 for PM
10
[50]
and 2.77 for PM
2.5
[49,51].
Statistical Analysis of Data
Results are presented as mean ±standard deviation
(SD) or median with range. Analysis between groups
was performed using Student’sttest, chi-square test, or
Mann–Whitney Utest, as applicable. The possibility of
an association between measured parameters with age,
BMI, family income, education, cooking hours per day,
lifetime duration of cooking (cooking years), kitchen
location, family size, number of smokers in the family,
use of mosquito repellant, and PM
10
and PM
2.5
levels in
cooking areas was tested using univariate regression
analysis. Variables that showed significant association
were later included in a backward stepwise multiple
regression model to adjust for their effects. Statistical
analyses were performed using EPI info 6 and SPSS
statistical software (Statistical Package for Social Scien-
ces for Windows, release 10.0, SPSS Inc., Chicago,
USA) and p<0.05 was considered significant.
RESULTS
Demography
Demographic and socioeconomic characteristics of
the study population are summarized in Table 1. The
LPG and BMF users were comparable with respect to
age, BMI, cooking years, hours of cooking per day,
family income, environmental tobacco smoke for the
presence of a smoker in the family, food habit, and use
of mosquito repellant. However, BMF users were less
educated (p<0.05 in Mann–Whitney Utest) and their
family income was significantly lower than that of their
neighbors who used to cook with LPG (p<0.05 in
Student’sttest). Moreover, 40.7% of BMF-using house-
holds did not possess a separate kitchen and they used to
cook in an area adjacent to the living room. In contrast,
only 15.3% of LPG-using households lacked a separate
kitchen (p<0.05 in chi-square test).
Particulate Pollutants in Indoor Air
The mean concentration of PM
10
in biomass-using
kitchen during cooking hours was 625±150 μg/m
3
(range,
479–1,556 μg/m
3
)incontrastto129±42μg/m
3
(range,
97–254 μg/m
3
) in LPG-using kitchen. The corresponding
PM
2.5
level was 312±85 μg/m
3
(range, 262–715 μg/m
3
)
and 77± 29 μg/m
3
(range, 46–117 μg/m
3
). During
noncooking hours, PM
10
and PM
2.5
concentrations were
204± 98 μg/m
3
(range, 142–311 μg/m
3
) and 82± 21 μg/m
3
(range, 66–126 μg/m
3
), respectively, in biomass-using
kitchencomparedwith93±41μg/m
3
(range, 45–145 μg/
m
3
) and 45± 22 μg/m
3
(range, 24–72 μg/m
3
) in LPG-using
kitchen. Thus, the particulate pollution levels of cooking
areas in biomass-using households were significantly
higher than those in LPG-using households during
cooking and noncooking times (p<0.001).
Hematology and Sputum Cytology
The total leukocyte count in peripheral blood was
significantly higher (p<0.05 in Student’sttest) in
BMF users compared with LPG-using controls (8.7±
0.72 vs. 5.2±0.83 × 10
3
/μl, respectively). Moreover,
BMF users had elevated number of total cells in
sputum (88.9±23.7 vs. 65.2 ±13.1 cells/hpf, p<0.05)
than controls. Blood and sputa of BMF users
contained more inflammatory cells like neutrophils
(blood, 5,867± 445 vs. 3,708 ±281 cells/μl, p<0.01;
sputum, 58.9±17.4 vs. 42.3 ±10.6 cells/hpf, p<0.01),
eosinophils (blood, 552± 63 vs. 300 ±48 cells/μl, p<
0.01; sputum, 1.7± 0.7 vs. 0.4 ±0.2 cells/hpf, p<0.01),
and lymphocytes (blood, 2,100± 244 vs. 1,929±207
cells/μl, p>0.05; sputum, 4.6± 1.3 vs. 3.3±1.0 vs.
cells/hpf, p<0.05).
Higher Surface Expression of CD16, CD35,
and CD11b/CD18 in Circulating Neutrophils
of Biomass Users
Flow cytometric analysis revealed significantly
higher MFI of CD16, CD35, CD11b, and CD18 in
circulating neutrophils of BMF users when compared
with that of LPG-using controls (p< 0.0001; Fig. 1). In
biomass users, the MFI of neutrophilic surface receptor
CD16 and CD35 was increased by 2.5-fold (3,196.1 ±
676 Banerjee, Mondal, Das, and Ray
Author's personal copy
337.5 vs. 1,282.1± 613.3 in controls) and 1.4-fold
(81.9±9.5 vs. 56.7 ± 4.2), respectively. Moreover, the
MFI of CD11b and CD18 on neutrophils was increased
by 37% and 66%, from 736.3±278.4 and 217.9± 93.1 in
controls to 1,008.2±234.3 and 362.1± 79.7 in biomass
users, respectively (Fig. 1).
Rise in Proinflammatory Cytokines and Chemokine
in Biomass Users
Tab le 2shows significantly elevated levels of
proinflammatory cytokines TNF-α, IL-6, and IL-12
and chemokine IL-8 in plasma of BMF users in
comparison with that of LPG users. Compared with
LPG-using control women, biomass users had 72%,
67%, and 54% higher plasma levels of TNF-α, IL-6, and
IL-12, respectively. Moreover, the plasma level of IL-8
was doubled in biomass users (Table 2).
Overexpression of iNOS and NO in Biomass Users
iNOS enzyme was found located in the cyto-
plasm of the neutrophils. Compared with LPG users,
the percentage of iNOS-expressing isolated blood
Table 1. Demographic and Socioeconomic Characteristics of BMF- and LPG-using Women
Variable LPG users (n=126) BMF users (n=142) pvalue
Age in years, median (range) 33 (24–41) 34 (23–40) NS
Median BMI (kg/m
2
) 24.3 23.9 NS
Cooking years, median (range) 13 (5–25) 14 (5–22) NS
Cooking hours per day, median (range) 3 (2.5–5.5) 3 (2.5–5.5) NS
Years of schooling, median (range) 8 (3–14) 3 (0–10) <0.05
Homes with separate kitchen (%) 84.7 59.3 <0.05
Smoker in family (%) 43.9 44.1 NS
Use of mosquito repellant at home (%) 75.8 74.1 NS
Food habit, mixed (%) 97.2 96.9 NS
Members in family, median (range) 4 (2–6) 4 (2–7) NS
Family income per month in US dollars (mean ±SD) 58.41± 8.32 31.78±7.17 <0.05
The LPG- and biomass-using groups were compared by chi-square test (for results presented as percentages), Mann–Whitney Utest (for median
values with range), and Student’sttest (for mean± SD) and p<0.05 was considered significant
nnumber of subjects, NS statistically not significant
Fig. 1. Histograms showing the MFI of surface expressions of CD16 (a), CD35 (b), and β
2
Mac-1 integrin (CD11b/CD18; c,d) on circulating
neutrophils of rural women who cooked exclusively either with biomass or LPG for the past 5 years or more. Bars represent the SD of the mean;
*p<0.0001, compared with the LPG users.
677Neutrophilic Inflammatory Response and Oxidative Stress
Author's personal copy
neutrophils was slightly increased in biomass users,
but the increment was not statistically significant (5.1
±1.8% vs. 4.8± 1.3%, p>0.05inStudent’sttest). In
airway neutrophils, however, iNOS expression was
increased three times over controls in women who
cooked exclusively with biomass (25.7± 8.4% vs. 8.9
±3.3% iNOS-expressing cells in sputum of control
women, p<0.0001). On the other hand, serum level
of NO was increased by twofold from 23.3±12.6 μM
in controls to 47.3±16.2 μMinbiomassusers
(Fig. 2).
Higher MPO Activity in Biomass Users
Spectrophotometric analysis revealed significantly
higher MPO activity in blood neutrophils of biomass
users than that of LPG-using control women (0.516 ±
0.10 vs. 0.2644±0.07 IU/ml, p< 0.001).
Oxidative Stress Among Biomass Users
Flow cytometric analysis showed a significant rise
in MFI of DCF-DA among BMF users, suggesting
increase in ROS generation in peripheral blood leuko-
cytes (neutrophils, monocytes, and lymphocytes) and
unfractionated sputum cells that contained alveolar
macrophages, epithelial cells, airway neutrophils, eosi-
nophils, and lymphocytes (Table 3). The MFI of DCF-
DA was 36% higher in peripheral blood neutrophils,
25% higher in monocytes, and 14% higher in lympho-
cytes of BMF users, relative to controls. ROS generation
was doubled in sputum cells of biomass users when
compared with that of controls (Table 3). On the other
hand, biomass-using women had 39% and 28% lower
levels of SOD in erythrocytes (156.8 ±65.2 vs. 255.1±
59.3 U/ml in control, p<0.0001) and sputum cells
(565.3±115.2 vs. 785.4±183.8 U/ml, p< 0.0001),
respectively, in comparison with controls.
ROS, SOD, and Neutrophil Activation
Spearman’s rank correlation analysis showed a
positive association between markers and mediators of
neutrophil activation and generation of ROS in biomass-
using women. Conversely, a negative association was
found between these markers and the level of antiox-
idant enzyme SOD (Table 4). However, the association
between IL-12 with ROS generation and SOD level was
not statistically significant (p>0.05).
Particulate Pollutants and Neutrophil Activation
In univariate analysis, levels of particulate pollu-
tants (PM
10
and PM
2.5
) in indoor air, family size, family
income, education, and kitchen location were positively
associated with different neutrophil activation bio-
markers. After controlling confounding factors in multi-
Table 2. Comparison of Proinflammatory Cytokine and Chemokine
Levels in Plasma of LPG and BMF users
Concentration in
plasma (pg/ml) LPG users (n=126) BMF users (n= 142)
TNF-α
Mean± SD 13.2±4.6 22.84±4.13*
Median (range) 11.8 (1.3–18.6) 21.1(17.9–29.5)**
IL-6
Mean± SD 28.5±8.6 47.5± 13.2*
Median (range) 27.5 (11.2–39.4) 43.9 (31–73.9)**
IL-12
Mean± SD 118.6± 48.65 183.05± 54.6*
Median (range) 114.1 (67.1–208.2) 174.8 (80.6–331.0)**
IL-8
Mean± SD 23.3±4.7 46.7±18.3*
Median (range) 19.6 (1.5–31.2) 31.5 (12.4–109.5)**
*p<0.0001, compared with the LPG users in Student’sttest; **p<0.001
in Mann–Whitney Utest
Fig. 2. Histograms showing comparison of the levels of iNOS in neutrophils (a,b) and NO in serum (c) between LPG and BMF users. Bars represent
the SD of the mean; *p<0.0001, compared with the LPG users in Student’sttest.
678 Banerjee, Mondal, Das, and Ray
Author's personal copy
variate logistic regression, a strong positive association
was observed between neutrophil activation biomarkers
with PM
10
and PM
2.5
as shown in Table 5.
Correlation Between Neutrophil Activation
Biomarkers and Years of Cooking with Biomass
A strong positive correlation was found in Spear-
man’s rank correlation test between years of cooking with
biomass and proinflammatory cytokine TNF-α(rho [ρ]=
0.9103, p<0.0001), IL-6 (ρ=0.8251, p<0.001), and IL-12
(ρ=0.6316, p<0.001), proinflammatory chemokine IL-8
(ρ=0.8797, p<0.0001), neutrophil surface receptor CD35
(ρ=0.9912, p<0.0001) and CD16 (ρ=0.9037, p<0.0001),
CD18 (ρ=0.7998, p<0.002), CD11b (ρ=0.8875, p<
0.0001), neutrophil iNOS (ρ=0.6247, p<0.012), NO (ρ=
0.5797, p<0.031), and MPO (ρ=0.9623, p<0.0001).
Moreover, there was a strong positive association
between oxidative stress and exposure years of cook-
ing with biomass because cooking years correlated
positively with generation of ROS in sputum cells, in
terms of MFI of DCF-DA (ρ=0.9738, p<0.0001), and
negatively with concentration of SOD in erythrocytes
(ρ=−0.8993, p<0.0001).
DISCUSSION
The objective of this study was to examine whether
cumulative inhalation of biomass smoke activates
neutrophil towards the pathway of inflammation via
oxidative stress in a group of rural premenopausal
women in India who are actively engaged in cooking
for the past 5 years of more. Significantly increased
number of inflammatory cells was found in blood and
sputa of biomass-using women than LPG users, suggest-
ing inflammation. This finding tempted us to examine
whether the inflammatory response was mediated via
overexpression of neutrophilic surface molecules, excess
proinflammatorycytokine and chemokine release, and
other neutrophil activation biomarkers like iNOS and
MPO. As anticipated, biomass-using women of this
study had elevated circulating levels of proinflammatory
cytokine TNF-α, IL-6, and IL-12 and proinflammatory
chemokine IL-8. Besides, their neutrophils expressed
more CD16, CD35, and CD11b/CD18 on their surface
compared with their age- and sex-matched neighbors
who used to cook with cleaner fuel LPG. Collectively,
these findings suggest that chronic exposures to biomass
smoke elicit inflammatory response through the upregu-
lation of proinflammatory mediators and neutrophil
adhesion molecules.
Table 3. Comparison of ROS Generation by Blood Leukocytes and
Sputum Cells between Biomass users and Control Women
MFI of DCF-DA (in arbitrary units)
LPG users
(n=126)
BMF users
(n=142)
Blood neutrophils, mean± SD 420.6 ±143.3 572.5± 136.5*
Blood monocytes, mean± SD 245.6±102.5 306.4± 124.8*
Blood lymphocytes, mean± SD 157.5± 72.7 179.4± 91.1**
Whole sputum, mean± SD 346.4± 188.1 708.8 ±274.9*
*p<0.0001 and **p< 0.05, compared with the LPG users in Student’s
ttest
Table 4. Spearman’s Rank Correlation Analysis to Test an Association
Between Oxidative Stress and Markers and Mediators of Neutrophil
Activation
Neutrophil activation
biomarkers
With ROS With SOD
ρvalue pvalue ρvalue pvalue
CD16 0.516 0.048* −0.740 0.003*
CD35 0.601 0.021* −0.529 0.022*
CD11b 0.552 0.023* −0.378 0.041*
CD18 0.802 0.001* −0.508 0.021*
TNF-α0.543 0.037* −0.448 0.020*
IL-6 0.561 0.031* −0.396 0.038*
IL-12 0.112 0.124 −0.291 0.102
IL-8 0.604 0.021* −0.683 0.008*
iNOS 0.681 0.012* −0.714 0.009*
NO 0.536 0.042* −0.385 0.040*
MPO 0.622 0.013* −0.462 0.016*
*p<0.05, considered as significant
Table 5. Multivariate Logistic Regression Analysis of the Association
between Neutrophil Activation Markers and PM
10
and PM
2.5
Levels in
Indoor Air Controlling Potential Confounders
Neutrophil activation
biomarkers
With PM
10
With PM
2.5
OR 95% CI OR 95% CI
CD16 1.25 1.03–1.52 1.35 1.12–1.65
CD35 1.24 1.02–1.47 1.44 1.18–1.76
CD11b 1.33 1.12–1.56 1.68 1.35–1.96
CD18 1.37 1.15–1.63 1.64 1.25–2.13
TNF-α1.29 1.06–1.57 2.50 1.25–3.66
IL-6 1.24 1.04–1.64 1.89 1.66–2.23
IL-12 1.11 1.11–1.20 1.23 1.25–1.56
IL-8 1.37 1.15–1.63 1.64 1.25–2.13
iNOS 1.37 1.15–1.63 1.64 1.25–2.13
NO 1.33 1.12–1.56 1.68 1.35–1.96
MPO 1.37 1.15–1.63 1.64 1.25–2.13
All the associations were positive and statistically significant
OR odds ratio, CI confidence of intervals
679Neutrophilic Inflammatory Response and Oxidative Stress
Author's personal copy
CD16 is widely expressed on the neutrophil surface,
and upregulation of this receptor contributes to disease
severity in sepsis [52]. On the other hand, CD35 is
responsible for controlling complement activation and the
binding of soluble immune complexes [33]. The engage-
ment of these receptors on the surface of neutrophils
stimulates neutrophil phagocytosis, degranulation, and
generation of ROS. Rise in blood leukocyte number,
increase in inflammatory cells in spontaneously expecto-
rated sputa, overproduction of ROS, and depletion of SOD,
as observed in biomass-using women of this study, are
consistent with inflammatory response [53–55]. In addition,
increased expressions of neutrophil surface receptors CD16,
CD35, and CD11b/CD18 among biomass users may
suggest stimulation of the immune defense. CD11b on the
neutrophil surface plays an important role in the adhesion
and migration of neutrophils from circulation into the
airways and in the activation of neutrophils [17,56]. Apart
from its involvement in phagocytosis of opsonized bacteria,
CD11b/CD18 contributes to neutrophil aggregation and
adhesion to the endothelial surface [26]. Activated neutro-
phils express β
2
-integrin (CD11b/CD18) on their surface
because it is required for their transmigration into the area of
inflammation. Neutrophil influx from blood to the airway
space is driven largely by IL-8, a potent neutrophil chemo-
attractant produced by airway epithelial cells. Biological
components of air pollution activate alveolar macrophages
to produce TNF-αwhich, in turn, stimulates airway
epithelial cells to produce IL-8. Therefore, upregulation of
CD11b/CD18 expression suggests accelerated migration of
neutrophils from the circulation to the tissues following
chronic exposures of the airways to biomass smoke. It is
important to mention in this context that smoking of tobacco
causes upregulation of CD11b/CD18 expression on neu-
trophil surface [57], increase in neutrophil retention in the
lung, and stimulation of granulocyte–macrophage colony-
stimulating factor that can modulate neutrophil function
[58]. If the upregulation of CD11b/CD18 on neutrophil
surface is perpetuated, however, it can lead to adverse heath
outcome. For instance, augmented expression of neutrophil
CD11b is common among patients with COPD [59].
Neutrophils of biomass users also generated an excess of
MPO, suggesting neutrophil activation following cumula-
tive biomass smoke exposures. MPO, a heme protein, is a
critical mediator in coronary atherosclerosis [60]. The high
level of MPO in plasma due to the activation of neutrophils
is an early event in acute myocardial infarction and
potentially precedes myocardial injury [60].
Increase in the expression of iNOS in neutrophils
and remarkable elevation in serum NO are other
significant observations among biomass users. NO is
an intracellular signal transmitter. Its excessive produc-
tion via iNOS and a subsequent oxidative stress reaction
are thought to be critically involved in the pathophysi-
ology of pulmonary sepsis [61]. Increase in iNOS
mRNA expression, iNOS activity, and NO secretion
have been recorded in alveolar macrophages in hyper-
oxia-induced lung injury in rat [62]. Conversely, sup-
pression of NO generation via downregulation of iNOS
by selective iNOS inhibitors protects from lung injury
[63,64].
The participants of this study were all never-
smokers and tobacco nonchewers, and exposure to
environmental tobacco smoke for the presence of
smoker in the family was similar among biomass and
LPG users. Therefore, it seems that neutrophilic inflam-
matory changes among biomass-using women were not
due to tobacco smoking or chewing habit. The villages
where the participants resided were far from the high-
ways and busy road traffic. Bicycle and cycle rickshaw
were the principal mode of transport in these villages
and there were no air-polluting industries within 5 km
radius. Thus, ambient air pollution levels in the study
areas seemed negligible. Besides, biomass and LPG
users were neighbors, so the impact of outdoor air
pollution was similar in these two groups. Therefore, the
difference in indoor air quality between biomass- and
LPG-using households was perhaps responsible for the
inflammatory changes among biomass users. But the
precise mechanism by which IAP elicits inflammation is
currently unknown. Combustion of biomass generates
organic chemicals which become adsorbed on the outer
surface of the respirable PM and get entry inside the
respiratory tract through inhalation. Inorganic compo-
nent of the PM, especially the transitional metals, can
directly stimulate epithelial cells to produce IL-8 [65]
and consequent neutrophilic inflammation in the lung
[66]. However, biomass smoke contains several other
pollutants such as volatile organic compounds toluene,
xylene, and benzene [67] which can elicit inflammation
following inhalation [68].
Oxidative stress among biomass users, as evident
from the rise in ROS generation and depletion of SOD,
is another potential mechanism of inflammation in these
women. Like biomass smoke, environmental tobacco
smoke and ambient (outdoor) air pollution also cause
oxidative stress and airway inflammation [69,70].
Inhaled PM can induce pulmonary and systemic inflam-
mation [71]resulting in ROS generation from inflam-
matory cells. In essence, the present study shows that
680 Banerjee, Mondal, Das, and Ray
Author's personal copy
sustained exposures to biomass smoke during daily
household cooking cause the upregulation of circulating
neutrophil surface receptor expressions facilitating their
transmigration to the airways causing inflammation in
women in their child-bearing age. Sustained inflamma-
tion may cause tissue damage leading to various diseases
including COPD and cardiovascular illness. In view of
these possibilities of great public health concern, efforts
should be made to reduce IAP from biomass burning by
improving kitchen ventilation, by introducing improved
cook stoves, and finally by switching to cleaner fuel
options.
ACKNOWLEDGEMENTS
The authors gratefully acknowledge the financial
support received from Central Pollution Control Board,
Delhi in carrying out this study.
Conflicts of interest statement. The authors declare
that there are no conflicts of interest.
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