Science of the Total Environment 438 (2012) 293–298

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Immune cells and cardiovascular health in premenopausal women of rural Indiachronically exposed to biomass smoke during daily household cooking

Anindita Dutta a,b,⁎, Purba Bhattacharya b, Twisha Lahiri b, Manas Ranjan Ray b

a College of Environmental Sciences and Engineering, Peking University, Beijing, Chinab Chittaranjan National Cancer Institute, Kolkata, India

H I G H L I G H T S

► Effect of chronic biomass smoke exposure on immunological profile was investigated.► Marked suppression in the total number of T-helper (CD4+) cells and B (CD19+) cells was observed.► Appreciable rise was documented in the number of CD8+ T-cytotoxic cells, Treg cells and CD16+CD56+ natural killer (NK) cells.► Immunological profile changed with the type of biomass fuel used for the cooking and with kitchen location.

⁎ Corresponding author at: International Senior RFellow, College of Environmental Sciences and Engineer100871, China. Tel.: +86 10 62754789; fax: +86 10 62

E-mail address: anidu14@gmail.com (A. Dutta).

0048-9697/$ – see front matter © 2012 Elsevier B.V. Allhttp://dx.doi.org/10.1016/j.scitotenv.2012.08.065

a b s t r a c t

a r t i c l e i n f o

Article history:Received 30 January 2012Received in revised form 6 August 2012Accepted 16 August 2012Available online 23 September 2012

Keywords:Biomass smokeTreg cellsLymphocytesMonocytesRural womenIndia

Changes in cells of the immune system are important indicators of systemic response of the body to airpollution. The aim of this study was to investigate the immunological changes in rural women who havebeen cooking exclusively with biomass for the past 5 years or more and compare the findings with womencooking exclusively with liquefied petroleum gas (LPG). We conducted a cross-sectional analysis of theassociations between indices of indoor air pollution (IAP) and a set of immune assays. Biomass users illustratedmarked suppression in the total number of T-helper (CD4+) cells and B (CD19+) cells while appreciable risewas documented in the number of CD8+ T-cytotoxic cells and CD16+CD56+ natural killer (NK) cells. Aconsistent finding among biomass users was rise in regulatory T (Treg) cells. Among biomass users, peripherallymphocyte subpopulations, Treg cells, and the number of typical monocytes (CD16−CD64+ cells), antigenpresenting types (CD16+CD64− cells) and plasmacytoid cells (CD16−CD64− cells) were found to be signifi-cantly altered in those who daily cooked with dung in comparison to wood and crop residue users (pb0.05).Biomass users who cooked in kitchens adjacent to their living areas had significant changes in peripherallymphocyte subpopulations, typical monocytes (CD16−CD64+) with high phagocytic activity and antigenpresenting monocytes (CD16+CD64−) against women who cooked in separate kitchens (pb0.01). Thisstudy has shown that women who cooked exclusively with biomass fuel had alterations in immune defensecompared with their neighbors who cooked with LPG.

© 2012 Elsevier B.V. All rights reserved.

1. Introduction

Cooking and wood burning are significant sources of indoor airpollution (IAP) in developing nations like India. IAP from biomasssmoke is a complex mixture of fine and ultrafine particles, carbonmonoxide (CO), oxides of nitrogen and sulfur, transitional metals,polycyclic aromatic hydrocarbons, volatile organic compounds andbioaerosols (Smith, 2000). These particles are capable not only ofactivating endogenous sources of local inflammation (Chang et al.,1990; Mukae et al., 2001) and oxidative stress within pulmonary tissue

esearch Scholar/Postdoctoraling, Peking University, Beijing760755.

rights reserved.

(e.g. immune cells; Brook, 2008), but also of stimulating productionand release of polymorphonuclear leukocytes (PMN) and monocytesfrom the bone marrow (Goto et al., 2004; Swiston et al., 2008), whichare key players in executing the immune functions.

Reports have shown that inhalation of toxic particles and gassesinduces lung epithelial cells and alveolar macrophages to generate arich milieu of inflammatorymediators, which in turn produce an inte-grated local lung and systemic inflammatory immune response (Hoggand van Eeden, 2009; Vardavas et al., 2010). These complex inflam-matory processes and changes in the immune system are crucial inthe pathogenesis of disorders like chronic obstructive lung disease(COPD), lung cancer, and atherosclerosis (Domagala-Kulawik, 2008).

We have previously shown that women chronically exposed tobiomass smoke have greater prevalence of hypertension, increasedplatelet activity, elevated levels of oxidized low-density lipoprotein

294 A. Dutta et al. / Science of the Total Environment 438 (2012) 293–298

and anti-cardiolipin antibodies and hence, they are predisposed toincreased risk of cardiovascular disease development (Dutta et al.,2011). Atherosclerosis is an inflammatory and thrombotic disease. Acrosstalk between platelets and lymphocytes, and a role of plateletsin enhancing development of T-effector cell have been mentionedby Gerdes et al. (2011). In addition, immune-alteration that mayoccur from exposure to cooking smoke may lead to greater suscepti-bility to infection with elevated titer of immunoglobulin E (IgE;Mishra et al., 1999). Changes in cells of the immune system areimportant indicators of systemic response of the body to air pollution.Against this backdrop, we attempt to investigate, in this study, theimmunological changes in rural women who have been cookingexclusively with biomass for the past 5 years or more. The findingswere compared with that of age-matched women from same localitywho cooked exclusively with liquefied petroleum gas (LPG). To thebest of our knowledge, this study on immune-toxicity due to cumula-tive exposure to biomass smoke is the first of its kind in India.

2. Materials and methods

2.1. Study design

We conducted a cross-sectional analysis of the associations be-tween indices of indoor air pollution (IAP) and a set of immune assays.All women gave informed consent before participation in this study.

2.2. Study population

Womenwere recruited from rural villages of four districts (Hooghly,Burdwan, Nadia and Birbhum) ofWest Bengal, an eastern state in India.A total of 819 apparently healthy, never-smoking premenopausal mar-ried women with regular menstrual cycle (28±2 days) were enrolled.Among the participants, 434 women (age 24–44 yr, median 36 year)cooked exclusively with solid, unprocessed biomass such as cow dungcake, wood, dried leaves, jute stick, hay and paddy husk. They weregrouped as biomass-users. The remaining 385 women, aged 25–44 yr(median 35 yr), cooked with cleaner fuel LPG and accordingly, theywere grouped as reference or control. Subjects were women who alsomet the criteria for measurement of immunologic outcomes. None ofthem were taking hormone replacement therapy in the preceding6 months, and had no history of invasive cancer, diabetes, cardiovascu-lar disease, or asthma.Womenwere eligible if they had no current seri-ous allergies, were not regular (two or more times/week) users ofaspirin or other non-steroidal anti-inflammatory medications, andwere not using corticosteroids or othermedications known to affect im-mune function (Shade et al., 2004).

2.3. Questionnaires and interviews

Information on demographics (age, education, habits, occupationof the participants, average family income, cooking hours per day,cooking-years, kitchen and fuel type, family), occupation of thespouse and environmental tobacco smoke (ETS) was collectedthrough personal interview using structured questionnaire. Thestudy protocol was approved by the Ethics Committee of ChittaranjanNational Cancer Institute, Kolkata.

2.4. Immune measures

Twelve-hour fasting blood samples were taken between 08:30and 09:30 h following strict blood-draw criteria (Boynton et al.,2007). Blood samples (5 ml) were collected in vacutainer tubes[Becton Dickinson (BD), USA] containing ethylene diaminetetraacetate,tri-potassium (K3-EDTA) as anticoagulant. All immune assays wereconducted at the Chittaranjan National Cancer Institute in the Depart-ment of Experimental Hematology.

2.5. Flow cytometric analysis of immune cells

The procedure of Fujimoto et al. (2000) was followed for studyingthe expression of surfacemarkers of the immune cells: CD4+ (T-helperlymphocyte), CD8+ (T-cytotoxic/suppressive lymphocyte), CD19+(B-lymphocyte), CD16+CD56+ (natural killer cell), CD4+CD25+(Regulatory T cells or Tregs), CD16+CD64+, CD16+CD64−, CD16−CD64+ and CD16−CD64− (monocyte subsets).

Whole blood samples were analyzed by flow cytometry within 3 hof blood collection. A 25 μl aliquot of whole blood was diluted with75 μl of phosphate buffered saline (PBS, pH 7.4) and the dilutedblood samples were incubated with 10 μl each of fluorescein isothiocy-anate (FITC)- and phycoerythrin (PE)-conjugated monoclonal anti-bodies (BD Pharmingen, USA) raised against human cells andisotype-matched negative controls for 30 min in the dark at roomtemperature. The erythrocytes were then lysed by incubating thesamples with 2 ml of RBC lysing solution (BD, USA) for 5 min atroom temperature. Thereafter, the cells were fixed with 0.5% parafor-maldehyde and 15,000 events were acquired and analyzed in a flowcytometer (FACS Calibur with sorter, BD, San Jose, CA, USA). Cellswere identified from their characteristic forward and side scatter pro-file on dot plots and gated.

Data acquisition and analysis of FL-1 (FITC) and FL-2 (PE) weredone using Cell Quest software (BD, USA). The relative proportion ofimmune cells was calculated from the statistical package of the CellQuest software from quadrant gate setting.

2.6. Measurement of indoor air pollution

The mass concentration of particulate matter with aerodynamicdiameter less than 10 and 2.5 μm (PM10, PM2.5, respectively) wasmeasured for three consecutive days in each household for 8 h perday (7.00–15.00 h) covering both cooking and non-cooking time byreal-time laser photometer (DustTrak™ Aerosol Monitor, model8520, TSI Inc., Shoreview, MN, USA). The procedure has been de-scribed in detail earlier (Dutta et al., 2011).

2.7. Statistical analysis of data

The results were statistically analyzed by Student's ‘t’ test,Chi-square test and ANOVA; and pb0.05 was considered significant.Logistic regression analysis of data was done by SPSS 10.0 (Chicago, IL,USA) statistical package. Spearman's rank correlation test (expressedas rho) was done to test the degree of correlation between exposureand possible outcomes. pb0.05 was considered as significant.

3. Results

3.1. Demographic characteristics of the participants

Demographic and socio-economic characteristics of biomass- andLPG-using women are compared in Table 1. It is apparent that the twogroups were well-matched with respect to age, body mass index(BMI), years and hours of cooking, passive smoking due to presence ofsmokers in the family, food habit, and number of familymembers. How-ever, they differed significantly (pb0.05) with respect to education,family income and presence of separate kitchen. Compared with con-trols, biomass users were less educated and had lower family income(pb0.05). Moreover, 41.9% biomass-using households lacked separatekitchen against 11.6% of LPG-using households (pb0.05; Table 1).

3.2. Fall in CD4+andCD19+cells but rise in CD8+andCD16+CD56+cells

Biomass users had reduced percentage and absolute number ofCD4+ T helper (Th) cells but greater percentage and absolute num-ber of CD8+ T-cytotoxic (Tc) cells in their circulation (Table 2). As a

Table 1Comparison of demographic characteristics of the participants.

Parameters LPG users(n=385)

Biomass users(n=434)

Percentage of women in each age group20–29 years 37.4 36.430–39 years 59.7 59.4≥40 years 2.9 4.3

Body mass index (kg/m2), median (range) 22.8 (21.3–24.1) 22.1 (20.9–23.9)Years of cooking (% women in each category)

5–9 years 31.9 32.910–14 years 55.3 55.215–19 years 9.3 9.120–25 years 3.5 2.8

Cooking hours per day, median (range) 3.0 (2.5–5.0) 3.5 (2.5–6.0)Homes with separate kitchen (%) 88.6 58.1*Smokers in the family (%) 50.4 51.7Years of schooling, median (range) 9 (1–14) 3 (0–8)**Food habit, mixed (%) 99.3 98.6Occupation of the participants

Household work only (%) 89.9 87.4Household+agricultural work (%) 10.2 12.6

Alcohol drinking habit (%) 0 0Members in family, median (range) 4 (2–6) 5 (3–8)Family income per month in US $(mean±SD)

84±16 41±6***

PM10 (μg/m3) 97±34 275±93***PM2.5 (μg/m3) 51±23 158±64***

Significant (pb0.05) compared with control in Chi-square test (*), Mann–Whitney Utest (**) and Student's t-test (***). LPG, liquefied petroleum gas.

Table 2Percentage and absolute number of leukocyte subsets.

Parameters LPG users(n=385)

Biomass users(n=434)

CD4+% 41.3±4.5 35.4±6.8⁎

Cells/μl 936±98 878±101⁎

CD8+% 28.9±4.7 31.4±6.1⁎

Cells/μl 656±73 779±122⁎

CD19+% 16.2±1.6 13.8±1.7⁎

Cells/μl 366±42 345±43⁎

CD16+CD56+% 13.6±1.4 19.4±2.8⁎

Cells/μl 308±37 482±45⁎

CD4+:CD8+ 1.4:1 1.1:1

CD16+CD64−% 1.2±0.6 2.0±1.1⁎

Cells/μl 2.0±0.5 4.9±1.7⁎

CD16+CD64+% 3.3±0.9 6.7±2.1⁎

Cells/μl 5.5±1.5 16.4±5.1⁎

CD16−CD64−% 20.4±3.2 27.5±3.6⁎

Cells/μl 33.9±6.2 67.4±8.9⁎

CD16−CD64+% 75.1±4.8 63.8±6.4⁎

Cells/μl 124.7±7.11 156.3±15.4⁎

Results are expressed as mean±SD.⁎ pb0.01 compared with LPG user by Student's t-test.

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result, the ratio of CD4+:CD8+ cells were reduced from 1.4:1.0 to1.1:1.0. Biomass-using women showed a small but marked declinein CD19+ B-lymphocytes and a significant 56% increase in the num-ber of CD16+CD56+ natural killer (NK) cells (Table 2).

3.3. Increase in regulatory T cells (Treg) in biomass users

Biomass users had a greater percentage and absolute number ofCD4+CD25+ regulatory T cells (Treg cells) compared to cleanerfuel that is LPG users (107 cells/μl vs. 42.2 cells/μl in controls). SinceTreg cells down-regulate immune response, an increase in their num-ber suggests altered immunity in women who daily cooked withunprocessed biomass.

3.4. Changes in monocyte subtypes

In biomass-using women, 70.5% of circulating monocyte expressedCD64 on their surface against 78.4% in LPG-users. Compared with con-trol women, biomass users registered a modest but significant fall inthe percentage of CD16−CD64+ cells (63.8 vs.75.1%, pb0.01) but a2-fold rise in CD16+CD64+ cells (6.7 vs. 3.3%, pb0.001). Of the 21.6%of CD64-negative monocytes in control women, 1.2% cells expressedCD16 and the remaining 20.4% monocytes were negative for bothCD16 and CD64. The percentages of these two subsets — CD16+CD64− cells and CD16−CD64− cells were increased in biomass users by67% and 35%, respectively (Table 2).

The changes in monocyte subsets among biomass users relative tocontrols becamemore apparent when absolute numbers of these cellsin circulation were calculated from absolute monocyte number andpercentage of each subset. Total number of CD64+ cells was in-creased from 130.2/μl in LPG-users to 172.7/μl in biomass users,showing an increment of 33%. The number of typical monocytes(CD16−CD64+) was increased by 25%, and a massive 3-fold risewas recorded in the number of CD16+CD64+ cells (monocyteswith both dendritic cell and monocytic functions). Similarly, thenumber of circulating monocytes with high antigen-presentingcapacity (CD16+CD64− cells) was increased by 2.5-fold in biomassusers and the absolute number of plasmacytoid monocytes (CD16−

CD64− cells) was doubled (67.4±8.9 vs 33.9±6.2/μl, pb0.001).In essence, significant rise in the absolute number of all the four circu-lating monocyte subsets was observed in women who cooked withbiomass when compared with that of LPG-using control.

3.5. Effect of lifetime biomass smoke exposure on immune cells

Chronic biomass smoke exposure can bring appreciable immuno-logical changes in exposed individuals. In agreement, Table 3 shows aremarkable fall in CD4+ cells and CD19+ cells, and a sharp rise inCD8+ and CD16+CD56+ cells in those biomass users who weremaximally exposed to biomass smoke (pb0.01). Linear elevation inthe level of Treg was also observed in peripheral blood among bio-mass users on the basis of exposure category (Table 3). Comparedto the women of lowest exposure category, 1.3-fold more (pb0.01)Treg cells was found in the circulation of those exposed to longest du-ration (category III). Similarly, women of highest exposure categoryhad a significant rise for all of the monocyte subtypes in their bloodwhen compared with other two lower groups by ANOVA (Table 3).

In Spearman's rank correlation test, negative correlations were foundbetween biomass smoke exposure and number of CD4+ (−0.212, p=0.045) and CD19+ (−0.238, p=0.035) cells, while the correlationswere positive for CD8+ (0.222, p=0.015) and NK (0.289, p=0.012)cells, Treg (0.286, p=0.045) and different monocyte subtypes(CD16+64−:0.215, p=0.040; CD16+64+:0.230, p=0.045; CD16−64+:0.196, p=0.050; CD16−64−:0.211, p=0.045).

After controlling potential confounders by multivariate logistic re-gression, significant association was found between lifetime smokeexposure and NK cells (OR=3.6, 95%CI, 1.03–12.9), and Treg (OR=2.5, 95%CI 1.07–12.9), but this was not so with other lymphocyte sub-sets; CD4+ cells (OR=2.1, 95%CI, 0.8–4.1), CD8+ cells (OR=2.7,95%CI, 0.9–6.5) and CD19+ cells (OR=1.6, 95%CI, 0.6–4.5).

Table 3Alterations in peripheral leukocyte subsets on the basis of exposure category.

Parameters Exposure category

I(n=154)

II(n=144)

III(n=136)

CD4+ cells/μl 913±102 870±97 845±91.5⁎

CD8+ cells/μl 733±112 783±128 820±121⁎

CD19+ cells/μl 366±47 350±39 320±42.7⁎

CD16+CD56+ cells/μl 462±53 474±46.4 512±41⁎

CD4+CD25+ cells/μl 94±11.2 107±21.3 121±34.6CD16+CD64− cells/μl 3.8±1.3 5±2.1 5.8±1.7⁎

CD16+CD64+ cells/μl 13±4.7 16.3±5.3 20.1±6.6⁎

CD16−CD64− cells/μl 59.4±7.7 66±9.1 77.2±8.2⁎

CD16−CD64+ cells/μl 144±13.9 159±15.7 167.5±14.6⁎

Results are expressed as mean±SD; values compared by ANOVA; exposure categories—Ib10,000, II=10,000–20,000, III>20,000 h-years.

⁎ pb0.01.

Table 5Association of lymphocyte subpopulations with different kinds of biomass.

Parameters Wood Agricultural residue Cow-dung

CD4+ cells/μl 1.4 (0.3–4.1) 1.8 (0.2–12.9) 2.2 (0.6–9.1)CD8+ cells/μl 1.2 (0.4–2.9) 1.5 (0.5–3.5) 2.6 (1.06–5.2)⁎

CD19+ cells/μl 1.8 (0.3–9.7) 2.1 (0.6–7.3) 3.4 (1.1–9.2)⁎

CD16+CD56+ cells/μl 1.6 (0.4–2.8) 1.9 (0.3–3.2) 2.7 (1.08–5.3)⁎

CD4+CD25+ cells/μl 1.0 (0.6–1.3) 1.2 (0.8–1.7) 1.7 (1.02–4.1)⁎

Odds ratio at 95% Confidence Interval.⁎ Indicates significance.

296 A. Dutta et al. / Science of the Total Environment 438 (2012) 293–298

3.6. Changes in leukocyte subsets in relation to the type of biomass usedfor cooking

Among biomass users of ruralWest Bengal, peripheral lymphocytesubpopulations, Treg cells, and the number of typical monocytes(CD16−CD64+ cells), antigen presenting types (CD16+CD64−cells) and plasmacytoid cells (CD16−CD64− cells) were found tobe significantly altered in those who daily cooked with dung in com-parison to wood and crop residue users (Table 4, pb0.05 analyzed byANOVA). However, we did not find any marked alteration forCD16+CD64+ cells in the circulation of those three biomass-usinggroups (Table 4).

Moreover, logistic regression analysis revealed a strong associa-tion of usage of cow-dung with CD8+ cells, CD19+ cells, CD16+CD56+ cells, and Treg cells (Table 5); but not so with CD4+ cells.Cooking with wood or agricultural residue however, did not showany significant association (Table 5). Our result may imply amongthree biomass types, cow-dung can cause maximum immuno-alterations due to high pollutant content.

3.7. Alterations in lymphocyte subpopulations in relation to kitchenlocation

Our findings demonstrate that location of kitchen might have alsoplayed a role in bringing immunological alterations. Table 6 showsthat biomass users who cooked in kitchens adjacent to their livingareas had significant changes in peripheral lymphocyte subpopula-tions, typical monocytes (CD16−CD64+) with high phagocyticactivity and antigen presenting monocytes (CD16+CD64−) againstwomen cooked in separate kitchens (pb0.01). However, no apprecia-ble change was revealed between the two groups with respect to Treg(Table 6), and the remaining two subsets of monocytes (Table 6).

Table 4Changes in leukocyte subsets in relation to biomass fuel types.

Parameters Wood(n=219)

Agricultural residue(n=176)

Cow-dung(n=39)

CD4+ cells/μl 898±111 874±105 862±98⁎

CD8+ cells/μl 761±100.1 771±126.4 813±120⁎

CD19+ cells/μl 367±46 344±42.7 322±51⁎

CD16+CD56+ cells/μl 465±39 478±45 506±49.3⁎

CD4+CD25+ cells/μl 99±8.0 109±11.7 115±12.1⁎

CD16+CD64− cells/μl 4.5±1.4 5±1.7 5.6±2.0⁎

CD16+CD64+ cells/μl 15.8±4.3 16.6±5.2 17.2±4.7CD16−CD64− cells/μl 65±7.7 69±9.3 72±8.6⁎

CD16−CD64+ cells/μl 151±15.6 158±14.7 161.2±15.0⁎

Results are mean±SD; values compared by ANOVA.⁎ pb0.05.

4. Discussion

This study has shown that women who cooked exclusively withbiomass fuel had alterations in immune defense compared withtheir neighbors who cooked with LPG. Biomass users illustratedmarked suppression in the total number of T-helper (CD4+) cellsand B (CD19+) cells while appreciable rise was documented in thenumber of CD8+ T-cytotoxic cells and CD16+CD56+ natural killer(NK) cells. Chronic exposures to biomass smoke were perhapsresponsible for these alterations. This hypothesis is supported byfindings from this and several other investigations. First, thechanges in lymphocyte subpopulation in biomass-using women ofthis study were significantly correlated with lifetime exposure tobiomass smoke (exposure-years). Second, studies conducted inlaboratory animals and human volunteers have shown that benzo(a) pyrene, and to other polyaromatic hydrocarbons, which are abun-dant in biomass smoke (Zhang and Smith, 1996), cause immunesuppression (NFHS, 1999). Third, sustained exposure to benzene,another major constituent of biomass smoke, causes alterations inT-cell subsets like fall in CD4+ cells and rise in CD8+ cells(Moszczyński et al., 1995; Irons et al., 2005). Fourth, exposure to partic-ulate matter (PM) reduces the percentage of CD4+ cells (Frampton etal., 2006). Fifth, air pollution increases the percentage of NK cells(Dostal et al., 2000) and PMs especially PM2.5 increase CD8+ and NKlymphocytes in peripheral blood of school children in a concentration-dependent manner even after adjusting for potentially confoundingfactors (Leonardi et al., 2000). Taken together, these reports stronglysuggest that the changes in lymphocyte subpopulation of biomassusing women were due to their sustained exposure to smoke emittedfrom burning biomass during cooking. It was further observed that reg-ular cooking with dung and in poorly ventilated adjacent kitchen couldalso bring appreciable alterations in peripheral lymphocyte subsets.

A consistent finding among biomass users was rise in regulatory T(Treg) cells. These cells belong to a T cell subset that can inhibit effec-tor T cell responses. In humans, Treg cells are usually recognized bytheir surface expression of CD4 and CD25, the α chain of IL-2 recep-tor. However, FOXP3, a member of the forked winged helix familyof transcription factors, is recognized as the exclusive marker ofTreg cells. These cells are important for maintaining immune

Table 6Changes in peripheral leukocyte subtypes in relation to kitchen location.

Parameters Women having separatekitchen(n=226)

Women having kitchenadjacent to living room(n=208)

CD4+ cells/μl 916±97 840±112⁎

CD8+ cells/μl 751±128.5 807±106⁎

CD19+ cells/μl 367±41 323±52.5⁎

CD16+CD56+ cells/μl 459±43.7 505±50⁎

CD4+CD25+ cells/μl 106±10.1 107±11.5CD16+CD64− cells/μl 4.5±1.9 5.2±1.5⁎

CD16+CD64+ cells/μl 16±5.3 16.8±4.7CD16−CD64− cells/μl 66.6±8.5 68±9.0CD16−CD64+ cells/μl 152±15.4 160±14.9⁎

Results are mean±SD; values compared by Student's t-test.⁎ pb0.01.

297A. Dutta et al. / Science of the Total Environment 438 (2012) 293–298

tolerance and protection from autoimmune diseases. They exert theirregulatory activity either by cell-to-cell contact or by the release ofsuppressive cytokines such as transforming growth factor (TGFβ)and interleukin-10 (IL-10; Gangal and Chowgule, 2009). Treg cellsdirectly or indirectly suppress effector cells of allergic inflammation,such as mast cells, basophils, and eosinophils, and contribute toremodeling in asthma and atopic dermatitis (Akdis et al., 2005).

Under strong chronic inflammatory conditions, the number ofTreg cells may not be sufficient to skew T cell differentiation. IL-4 pro-duced by TH2 cells may abrogate the function of Treg cells by inducingthe formation of T cells producing IL-9 and IL-10 with no regulatoryproperties (Gangal and Chowgule, 2009). Treg cells could promotethe development of TH17 cells which downregulates production ofdendritic cells-derived TH2 chemo-attractant (Schnyder-Chandria etal., 2006). Therefore, an imbalance between TH2 and Treg cells couldresult in atopic diseases. In view of this, rise of Treg cells may indicatea defense against inflammatory manifestations and chronic infectionin biomass users.

Monocytes (Mo) are important for nonspecific defense againstpathogenic organisms. They exert immunoregulatory functions viacytokine production. Human peripheral blood Mo is a heterogeneouscell population differing in phenotype and immunoregulatory func-tion. Based on the expression of Fc gamma receptor I (FcγRI; CD64)and Fc gamma receptor III (FcγRIII; CD16), monocytes can beclassified into four categories (Grage-Griebenow et al., 2000). Com-pared with LPG users, biomass using women showed a significantrise in the absolute number of all four circulating monocyte subsets.CD16−CD64− cells are plasmacytoid Mo and CD16−CD64+ cellsare typical Mo that are highly phagocytic. CD16+CD64+ cells havevaried functions. Like dendritic cells, they stimulate T-cells to induceTH1 responses. In addition, they are highly phagocytic that produceintracellular superoxide anion (Grage-Griebenow et al., 2000). Be-cause of their novel characteristics of both monocytes and dendriticcells, CD16+ CD64+ are important for innate as well as adaptive im-mune responses. On the other hand, CD16+CD64− monocytes arelow in phagocytic activity and cytokine production, but highlyexpress MHC class I, class II and costimulatory B7 molecules and in-duce adaptive TH1 responses against bacterial and viral infections. Inview of these, increase in all of the Mo subsets may indicate systemicinflammation and infection in biomass fuel users.

β2 Mac-1 integrin is expressed on the surface of many leukocytessuch as monocytes, granulocytes, macrophages, and natural killercells. It mediates inflammation by regulating leukocyte adhesionand migration and has been implicated in several immune processessuch as phagocytosis, cell-mediated cytotoxicity, chemotaxis andcellular activation (Solovjov et al., 2005; Schymeinsky et al., 2007). Itis involved in the complement system due to its capacity to bindwith inactivated complement component 3b (iC3b) (Arnaout et al.,1983). The expression of this integrin on activated leukocyte plays apivotal role in defense against invading pathogens as its ligands includethe complement fragment iC3b, intercellular adhesive molecule-1(ICAM-1; CD54), fibrinogen and bacterial lipopolysaccharide, i.e. LPS(Nicholson et al., 2007). The importance of this integrin in the host de-fense can be judged by the report that showed facilitation of bacterialgrowth following blockade of leukocyte CD11b/CD18 (Nilsson et al.,2005). Besides, Mac-1 integrin molecules mediate antigen presentationand/or T cell activation (Sandilands et al., 2005). Sustained over-expression of Mac-1, however, could be harmful because it prolongsinflammation by delaying neutrophil apoptosis leading to tissue dam-age (Yan et al., 2004). Overexpression of Mac-1 integrin on the surfacesof both neutrophils and monocytes of biomass-using women of thisstudy can be viewed against this perspective. In addition, significantpositive correlations were recorded for lifetime smoke exposure withCD11b and CD18 on circulating neutrophils and monocytes. Hence, itmay be inferred that inflammatory responses among solid fuel userscan also be, at least in part, contributed by upregulation of CD11b/

CD18 complex due to chronic exposure to toxic pollutants present im-mensely in biomass smoke. Moreover, women who cooked solely withunprocessed biomass had a higher risk for cardiovascular illness as arise in CD11b expression has been linked to activation of coagulationcascade and endothelial perturbation (Falanga et al., 2000).

5. Conclusions

This study has shown that indoor air pollution from biomass fueluse is a serious health hazard for rural people in eastern India.Womenwho cook with these fuels are the worst hit. This is a neglectedarea of public health that affects the majority of nation's population. Inview of the enormity of the problem, the issue of indoor air pollutionshould be dealt seriously. Efforts should be made by all concerned tounderstand the problem better and to formulate strategies to reduceemission and exposure to biomass smoke. We recommend introduc-tion of improved cook stoves with increased combustion efficiency,and improvement on kitchen ventilation through installation ofchimneys, and ultimately switch to cleaner fuel such as LPG and naturalgas.

Acknowledgment

The study was funded by the Council of Scientific and IndustrialResearch, India and the Central Pollution Control Board, Delhi underthe Ministry of Environment and Forests, Government of India.

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