Inhalation Toxicology, 17:161–168, 2005Copyright c© Taylor & Francis Inc.ISSN: 0895-8378 print / 1091-7691 onlineDOI: 10.1080/08958370590904616

Proinflammatory and Anti-Inflammatory CytokineBalance in Gasoline Exhaust Induced Pulmonary Injuryin Mice

Veerapandian SureshkumarInhalation Toxicology Laboratory, Industrial Toxicology Research Center, Mahatma Gandhi Marg,Lucknow, India

Bholanath PaulImmunobiology Laboratory, Industrial Toxicology Research Center, Mahatma Gandhi Marg,Lucknow, India

Mani UthirappanInhalation Toxicology Laboratory, Industrial Toxicology Research Center, Mahatma Gandhi Marg,Lucknow, India

Renu PandeyImmunobiology Laboratory, Industrial Toxicology Research Center, Mahatma Gandhi Marg,Lucknow, India

Anand Prakash SahuPreventive Toxicology, Industrial Toxicology Research Center, Mahatma Gandhi Marg,Lucknow, India

Kewal Lal, Arun Kumar PrasadInhalation Toxicology Laboratory, Industrial Toxicology Research Center, Mahatma Gandhi Marg,Lucknow, India

Suresh Srivastava, Ashok SaxenaImmunobiology Laboratory, Industrial Toxicology Research Center, Mahatma Gandhi Marg,Lucknow, India

Neeraj MathurEpidemiology Division, Industrial Toxicology Research Center, Mahatma Gandhi Marg,Lucknow, India

Yogendra Kumar GuptaDirector, Industrial Toxicology Research Center, Mahatma Gandhi Marg, Lucknow, India

Received 5 May 2003; accepted 24 October 2004.This work was carried out as an in-house project. The help rendered by Dr. S. K. Bhargava, Dr. S. Barman, and Dr. G. C. Kiskhu, of the

Environmental Monitoring Group, in evaluating the CO, SOx, and NOx concentration in the gasoline exhaust is gratefully acknowledged. Wealso acknowledge the technical assistance rendered by Sri B. K. Maji, Sri Ram Kumar, and Shiv Pyare during the entire course of the work. Thisis ITRC manuscript number 2277.

Address correspondence to B. N. Paul, Immunobiology Laboratory, Industrial Toxicology Research Center, PO Box 80, M.G. Marg, Lucknow226001, India. E-mail: bholanathpaul@yahoo.com

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162 V. SURESHKUMAR ET AL.

Proinflammatory and anti-inflammatory cytokine balance and associated changes in pulmonarybronchoalveolar lavage fluid (BALF) of unleaded gasoline exhaust (GE) exposed mice were in-vestigated. Animals were exposed to GE (1 L/min of GE mixed with 14 L/min of compressedair) using a flow-past, nose-only, dynamic inhalation exposure chamber for different dura-tions (7, 14, and 21 days). The particulate content of the GE was found to be 0.635 ± 0.10 mgPM/m3. Elevated levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) were ob-served in BALF of GE-exposed mice, but interleukin 1β (IL-1β) and the anti-inflammatorycytokine interleukin-10 (IL-10) remained unaffected. GE induced higher activities of alkalinephosphatase (ALP), γ-glutamyl transferase (γGT), and lactate dehydrogenase (LDH) in theBALF, indicating Type II alveolar epithelial cell injury, Clara-cell injury, and general toxicity,respectively. Total protein in the BALF increased after 14 and 21 days of exposure, indicatingenhanced alveolar-capillary permeability. However, the difference in the mean was found statis-tically insignificant in comparison to the compressed air control. Total cell count in the BALF ofGE-exposed mice ranged between 0.898 and 0.813 × 106 cells/ml, whereas the compressed aircontrol showed 0.65 × 106 cells/mL. The histopathological changes in GE-exposed lung includesperivascular, and peribronchiolar cuffing of mononuclear cells, migration of polymorphonu-clear cells in the alveolar septa, alveolar thickening, and mild alveolar edematous changesindicating inflammation. The shift in pro- and anti-inflammatory cytokine balance and eleva-tion of the pulmonary marker enzymes indicate toxic insult of GE. This study will help in ourunderstanding of the mechanism of pulmonary injury by GE in the light of cytokine profiles,pulmonary marker enzymes, and lung architecture.

Vehicular pollution is one of the most significant contribu-tors to urban air pollution in recent time. Inhalation is one of themost important routes of human exposure to various automo-bile exhausts and results in exacerbation of illness from respira-tory diseases (Zanobetti et al., 2000). It has been estimated thatemission from diesel and gasoline powered vehicles can con-tribute 20–40% to the ultrafine aerosol organic carbon emissionand its contribution is still increasing (Hildemann et al., 1991),but their real impact on health has not been fully elucidated(Marano et al., 2002). The exhaust of both gasoline and dieselengines contains carbon monoxide (CO), hydrocarbon (HC),carbon dioxide (CO2), oxides of nitrogen (NOx), and particu-late matter (PM). However, it is true that the chemistry of gaso-line exhaust (GE) is quite different from that of diesel exhaust(DE) due to the fact that DE contains heavier hydrocarbons,C10–C22, whereas GE components typically range from C1 toC12. It is also true that DE in comparison to GE has low CO, HC,and CO2, and high NOx and PM (Central Pollution and ControlBoard, 2001). Hydrocarbons from C12 to C22 are too heavy toremain in the gaseous state at room temperature and thereforepose a different situation in terms of pollution and sampling(Smith, 2002). These quantitative differences may have an im-pact on the severity of the exposure effect, but the problempersists.

Although considerable research has been directed toward thehealth risks of diesel exhaust during the past 20 years, gasolineexhaust has not been studied nearly as extensively (Mauderly,1994). The fact that pollution associated with engine exhaustmay contain a substantial contribution from gasoline engines isoften overlooked. However, gasoline exhaust particulate matteris known to contain a variety of mutagenic and carcinogenicagents, including polycyclic aromatic hydrocarbons, which areformed by incomplete combustion of fossil fuel (Westerholmet al., 1988).

The general pathological response to an inhaled toxicantis said to be epithelial cell injury and the triggering of acuteinflammatory–immune processes (Cross et al., 1997). The ep-ithelial cells of the airway and alveoli encounter particles afterinhalation, which results in a number of respiratory diseases(Baeza-Squiban et al., 1999). The traditional view of the ep-ithelium as a relatively passive physical barrier to the externalenvironment has been superceded by the concept that the ep-ithelial cell plays a key role in regulating airway inflammation(Polito & Proud, 1998; Refsnes et al., 2001). Epithelial cellslining respiratory airways can act as “effector” cells (Cohn &Adler, 1992) and participate in inflammation in number of ways.They can act as target cells, responding to exposure to a vari-ety of inflammatory mediators and cytokines by altering one orseveral of their functions such as mucin secretion, ion transport,or ciliary beating. Aberrations in any of these functions can af-fect local inflammatory response and compromise pulmonarydefense (Adler et al., 1994).

In view of this, we assess the pulmonary injury by GE andthe proinflammatory and anti-inflammatory cytokine balancein the bronchoalveolar lavage of GE-exposed mice.

MATERIALS AND METHODS

AnimalsHealthy male Swiss mice (20–25 g body weight, 10–12 wk

age) obtained from GLP compliant Animal Breeding Facil-ity of Industrial Toxicology Research Center, Gheru Campus,Lucknow, were acclimatized for 1 wk prior to inhalation expo-sure by keeping the mice in mice-holding tubes for 7 days fora duration of 15 min/day. These mice were maintained understandard conditions of husbandry (22 ± 2◦C room temperatureand 50–55% relative humidity) and were provided pelleted feed(Nav Maharastra Chakan Oil Mills, India) and water ad libitum.

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CYTOKINES IN MICE EXPOSED TO GASOLINE EXHAUST 163

The entire experiment was performed with the approval of theInstitutional Ethics Committee.

Inhalation Exposure to Gasoline ExhaustThe animals were divided into four groups of six mice each.

The exposure to unleaded gasoline exhaust was carried out ina flow-past, nose-only, dynamic inhalation exposure chamber(Intox Products, Albuquerque, NM) for 15 min/day for 7, 14,and 21 days in separate groups. The exposure duration wasadopted from Lall et al. (1998). Exposure timing remained con-sistent for each day during the light phase of the light:darkcycle throughout the experiment. Animals exposed to com-pressed air for 21 days served as control. Compressed air wasgenerated by a compressed air motor (In Tox Products, USA;model 1023-0131Q-G608X) containing an air-filtering deviceand ultimately sent through a compact air dryer (Gem Airdryer equipment, India). A Genset (Honda portable generator,EBK-1200, four-stroke, one-cylinder-engine type, Honda SpielPower Products Limited, India) was used to generate GE frompetrol fuel typically used for automobiles. Unleaded gasolinewas procured in bulk from a local filling station at Lucknow,India, and stored in a refrigerator (4◦C) in an airtight aluminumcontainer. The emission from the generator was passed throughcopper tube coils of 5 m length via a glass chamber of 21 Lcapacity connected to a rotameter and diluted with compressedair in the ratio of 1:14 before being introduced into the noseonly inhalation exposure chamber (Figure 1). This dilution ra-

FIG. 1. (a) Schematic diagram of the gasoline exhaust inhala-tion exposure system. Arrows indicate direction of the airflow.(b) Detailed diagram showing the inhalation chamber with amouse holding tube showing the central plenum of inhalationchamber, negative pressure zone, and location of apertures inmouse holding tube.

tio closely represents the total suspended particle (TSP) con-centration at the capital city, Delhi (TSP 0.409 ± 0.11 mg/m3

in 1997 and 0.365 ± 0.1mg/m3 in 1998) (Aneja et al., 2001).The copper tube coils helped to reduce the temperature of theexhaust. The flow rate in the gasoline exposure chamber was15 L/min and the pressure in the control and GE exposure cham-ber was −0.5 in of water as indicated in the control panel ofthe exposure system. A 25-mm in-line filter holder (Intox Prod-ucts, Albuquerque, NM) was fitted with the inhalation chamberfor drawing air through a glass-fiber filter paper (Type A/E,25 mm, Gelmen Sciences, Inc., Ann Arbor, MI) for collectionof GE particulates. The GE particulates collected were mon-itored by measuring the change in weight of glass-fiber filterpaper and calculated via the methodology adopted elsewhere(Lall et al.,1998). The particulate content of the GE was foundto be 0.635 ± 0.10 mg PM/m3. The particle size profile of GEwas measured with the help of cascade impactor (In-Tox Prod-uct, Albuquerque, NM). GE was found to contain particle ofsizes ranging from >4,<4 to 3,<3 to 2, <2 to 1.5, <1.5 to 0.5,and <0.5 µm particles in the ratio of 34.17, 15.78, 15.78, 10.57,5.29, and 18.39%, respectively. The chamber concentrations ofSO2, NOx, and CO were found to be 0.11 mg/m3, 0.49 mg/m3,and 18.67 ppm, respectively. SOx and NOx were measured bycalorimetric methods (Indian Standard, 2001; Indian Standard,1975), and CO level was monitored by using a hand-held COmonitor (Riken-Keiki, Japan). The entire inhalation exposureequipment was operated at 22 ± 2◦C ambient temperature withrelative humidity ∼50%. The mouse holding tubes had 4 aper-tures spaced at 90◦ angle near the nose portion of the tube thatopens in to the negative pressure zone of the inhalation chamber(Figure 1b). These apertures helped in removing the exhaled airand unused GE to prevent accumulation in the mouse holdingtubes. Furthermore, the dryness of the body hair of mice after15 min of exposure apparently indicated the presence of a fairlybios environment in the chamber. Mice, however, kept for >1 hin the chamber showed wetness in the body hair, indicatingsignificant increase in the relative humidity inside the chamber.

Collection of Bronchoalveolar Lavage FluidAfter completion of 7, 14, and 21 days of GE exposure,

the mice from the respective groups were anesthetized by in-jecting sodium thiopentone, ip (5 mg/0.1 ml/mice) within 1 hafter the last exposure for collection of bronchoalveolar lavagefluid (BALF). The trachea was exposed by midline incision inthe neck region and 1 ml of phosphate-buffered saline, pH 7.4(PBS), was injected into the lungs through trachea and with-drawn after 10 s. The recovery of BALF ranged from 70 to80% of the volume instilled. BALF from each mouse was cen-trifuged at 2000 rpm, for 5 min, at 4◦C, and the supernatantwas subjected to cytokine and enzyme analysis. The pelletedcells were subjected to total cell count by using a Neubauerchamber and differential counts based on Giemsa staining. Theviability of the cell was measured by trypan blue dye exclusiontest.

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164 V. SURESHKUMAR ET AL.

Cytokine AnalysisA solid-phase sandwich enzyme-linked immunosorbent as-

say (ELISA) kit (R & D Systems, USA) was utilized for theevaluation of tumor necrosis factor-α (TNF-α), interleukin-6(IL-6), interleukin-1β (IL-1β), and interleukin-10 (IL-10). Theprotocol reported elsewhere by us has been adopted (Paul et al.,2002).

Biochemical AnalysisThe enzyme activities of alkaline phosphatase (ALP) and

γ -glutamyl transferase (γ GT) were assayed using an autoan-alyzer (Chemwell, Awareness Technology, USA). Lactate de-hydrogenase (LDH) activity and protein contents of the BALFsupernatant were assayed according to the methodologies ofVassault (1983) and Lowry et al. (1951), respectively.

HistopathologyLungs were harvested on days 7, 14, and 21 after expo-

sure to GE and inflated by gentle injection of 4% phosphate-buffered formalin fixative until they reached approximately nor-mal anatomic volume. The pressure required for inflammationwas not measured but was judged to be similar in all animals.Cubes of lung tissue were removed after several minutes fixa-tion in situ, gently agitated in the fixative at room temperaturefor 24 h, and then washed and stored in 70% ethanol fixative.Tissue was embedded in paraffin, sectioned, and stained withhematoxylin and eosin (Sahu & Lal, 1995).

Statistical AnalysisOne-way analysis of variance (ANOVA) was performed to

find out significant differences in mean values of the parame-ters at different time points using EPI-INFO 5.0 software afterascertaining the homogeneity of variance between the differentgroups. Post hoc analysis was done to compare pairwise themeans of different exposure time periods from control us-ing Dunnett’s testing procedure (Zar, 1984). For parametersshowing heterogeneity, nonparametric Kruskal–Wallis one-way analysis of variance was performed using EPI-INFO 5.0software.

RESULTS

Effect of GE Exposure on CytokinesThe background level of proinflammatory cytokines TNF-α,

IL-6, and IL-1β in the BALF was 20.2 ± 4.01, 7.65 ± 0.77,and 15.25 ± 1.7 pg/ml, respectively, and that of the anti-inflammatory cytokine IL-10 was 163.3 ± 23.1 pg/ml. GE ex-posure in mice has been found to modulate the proinflammatorycytokines like TNF-α and IL-6 in the BALF on days 14 and 21in comparison to both compressed air control and 7-day GEexposure. In contrast, IL-1β was found unaffected through outthe course of the study (Figure 2). Seven days of GE exposuredid not show any change in the TNF-α, IL-6, and IL-1β cy-tokines in comparison to compressed air control. IL-10 level in

FIG. 2. Pro- and anti-inflammatory cytokine profile in BALFof GE-exposed mice. Data represents mean ± SE of valuesfrom six mice in each group. Asterisk indicates statisticallysignificant, p < .001. TNF-α; IL-6; � IL-10, and �IL-1β.

the BALF of GE exposed mice was found unaffected at all thetime points.

Effect of GE Exposure on Marker Enzymes of Lung InjuryWe observed significantly increased activities of ALP,

γ -GT, and LDH in BALF of 14- and 21-days GE-exposedmice in comparison to compressed air exposed control and7-days GE-exposed animals (Table 1). ALP is a membrane-bound enzyme of pulmonary type II cells along with surfactant(Henderson et al., 1995), and therefore increased activity inBALF may reflect damage of pulmonary type II cells. Similarly,Clara cells are reported to be rich in γ -GT, and enhanced γ -GTin BALF may be an indication of Clara-cell damage (Dinsdaleet al., 1992). The possibility of cells other than pulmonary typeII cells and Clara cells contributing to the ALP and γ -GT pool,respectively, in the BALF also remains. LDH activity representsgeneral cytotoxicity, and hence elevated activity of this enzymein the BALF is indicative of lung injury by GE. However, theelevation in the ALP, γ -GT, and LDH activities on day 21 is insignificant in comparison to 14-day GE exposure, suggestingthat maximum pulmonary injury takes place on day 14, afterwhich it attains a plateau. Total protein in BALF of 14- and21-days GE-exposed mice was found marginally increased incomparison to compressed air control but the differences wasstatistically insignificant (Table 1).

Total Cell Content in BALFThe leukocytes recovered from the lavage fluid showed mean

cell count ranging from 65 × 104 to 89.8 × 104 cells/ml

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CYTOKINES IN MICE EXPOSED TO GASOLINE EXHAUST 165

TABLE 1Effects of gasoline exhaust (GE) inhalation on lung biomarkers in BALF of mice

Groups γ -GT (U/L) ALP (U/L)LDH (nmole

NADH oxidized/min)Total

protein (mg/ml)Total cell

count (106/ml)

Compressed air exposed 1.67 ± 0.33 7.67 ± 1.1 40.98 ± 7.13 0.282 ± 0.007 0.65 ± 0.0827 days GE exposed 2.17 ± 0.48 8.5 ± 1.73 48.18 ± 3.63 0.3098 ± 0.011 0.898 ± 0.09814 days GE exposed 13.0 ± 4.84a 14.33 ± 1.96a 98.63 ± 15.59b 0.559 ± 0.124 0.813 ± 0.0921 days GE exposed 13.67 ± 3.6a 13.67 ± 1.8a 115.49 ± 7.0b 0.3127 ± 0.0062 0.855 ± 0.11

Note. Data represent mean ± SE of values from six mice in each group.aStatistically significant, p < .05.bStatistically significant, p < .01.

(Table 1). More than 95% of the cells were found viable inthe BALF of all the GE-exposed groups and, compressed aircontrol group. The leukocyte numbers in the BALF after GEexposure were found to be higher on days 7, 14, and 21 in com-parison to the compressed air control, but the difference failedto reach statistical significance. The differential count of thelavage cells in compressed air control group was 96.67 ± 0.61%macrophages, 0.83 ± 0.17% neutrophils, 1.67 ± 0.33% lym-phocytes, and 0.83 ± 0.40% eosinophils. The macrophage, neu-trophils, lymphocytes and eosinophils distribution in the BALFof 7 day GE-exposed mice was found to be 96.33 ± 0.56,1 ± 0.26, 1.83 ± 0.31, and 0.83 ± 0.31% respectively. Interest-ingly, the only notable change was observed in the neutrophil

FIG. 3. Histopathological examination of lung of mice exposed to GE for different duration. (A) Control mice lung showingnormal architecture of bronchoalveolar region. (B) 7-Days GE-exposed mice lung showing almost normal architecture of bron-choalveolar region except a few mononuclear cells found in the peribronchiolar and alveolar region. (C) 14-Days GE-exposedmice lung showing peribronchiolar and perivascular cuffing of inflammatory cells (→) and mild edematous changes in the alveolarregion (∗). (D) 21-Days GE-exposed mice lung showing sloughing of lining epithelial cells in the bronchiolar region (→). Focalaccumulation of mixed inflammatory cells in the alveolar region & slight thickening of alveolar septa ( →→ ). H&E, ×200.

level (>1%) (p > .05). The differential distributions of leuko-cyte population in BALF of 14- and 21-days GE-exposed micewere very similar to the 7-days GE-exposed group (not shown).

Histological ObservationThe histopathological examinations of lungs of compressed

air control mice showed normal pulmonary architecture with afew or no inflammatory cells in the alveolar lumen (Figure 3A).In 7-days GE-exposed mice, the lung histopathology did notshow any significant alteration as compared to the controlexcept for a few mononuclear cells in the peribronchiolar andalveolar region (Figure 3B). After 14 days of GE-exposure,perivascular and peribronchiolar cuffing of mononuclear cells

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166 V. SURESHKUMAR ET AL.

and mild edematous changes in the alveolar region were visi-ble (Figure 3C). Lungs of 21-days GE-exposed mice showedsloughing of epithelial cells lining the bronchiolar region andfocal accumulation of mixed inflammatory cells in the alveolarregion. Edematous changes in the alveolar region and thicken-ing of alveolar septa due to migration of inflammatory cells intothe interstitial space indicate mild inflammation (Figure 3D).

DISCUSSIONIn this study, we observed that GE exposure causes injury in

the respiratory tract of mice involving mediators, cells and as-sociated enzymes. Analyses of BALF in mice for componentsof proinflammatory cytokines, IL-1β, IL-6, and TNF-α haverevealed that IL-6 and TNF-α play an active role in lung in-flammation by GE, as significantly elevated levels of these twocytokines were reflected from day 14 onward following GE ex-posure. It is not clear why IL-1β was unaffected at all the timepoints following GE exposure, and this therefore warrants morestudies on this aspect. A study to assess the murine pulmonaryinflammation following instillation of ambient particulate mat-ter revealed increases in albumin and TNF-α with a high doseof fine particles (1.7 to 3.5 µm), while IL-6 levels were foundabove control levels with all the coarse, fine, and ultrafine parti-cles. Il-1β was not reported (Dick et al., 2003). Elevated levelsof cytokines that are pro-inflammatory in nature are consid-ered to orchestrate pulmonary damage (Li et al., 1995; Devlinet al., 1994; Martin et al., 1997). Therefore, elevation of IL-6and TNF-α bears significance in inducing pulmonary injury fol-lowing GE exposure. Importantly, GE exposure had no effecton the anti-inflammatory cytokine, IL-10, in the BALF duringthe entire course of our study, although elevation of proinflam-matory cytokines (TNF-α and IL-6) occurred. Alternatively, ashift in the balance between the pro- and anti-inflammatory cy-tokines occurred in the BALF of GE-exposed mice, favoringinflammation.

The cellular changes during pulmonary inflammation in-clude activation of alveolar macrophages and influx of poly-morphonuclear neutrophils (Perez et al., 1992; Brain, 1992).In the present study, a marginal increase but statistically in-significant cell content in the BALF of GE-exposed mice wasobserved in comparison to the compressed air control. Possiblythe gentle method of lavage adopted by us led to disproportion-ate recovery of cells from the airways. The gentle method oflavage was adopted to detect an inflammatory response withlittle or less disturbance to the lung. Differential leukocyteprofile in the GE-exposed groups revealed a marginal change(>1%) in the neutrophil percentages in comparison to com-pressed air control (<1%). The total protein content in BALFof 14- and 21-days GE-exposed mice was found elevated butstatistically insignificant. It has been reported that neutrophilinflux plays a major role in increasing the permeability of thealveolar-capillary barrier and providing cellular toxicity dur-ing inflammatory response (Henderson & Belinsky, 1993). Inour study we observed marginal increase in the neutophil pop-

ulation that led to marginal increase in the alveolar-capillarypermeability and may be the reason for slight but statisticallyinsignificant rise in total protein. The histopathological changeswere also mirrored in the GE-exposed lung, with the demonstra-tion of mild alveolar thickening, migration of polymorphonu-clear cells in the alveolar septa, and the alveolar edematouschanges suggesting a delayed effect and that the lungs are atrisk following GE exposure. This is because of the complexnature of the edema formation which involves alterations of themicrocirculation and accumulations of inflammatory cells, ini-tiated by resident macrophages that secrete pro-inflammatorycytokines in response to tissue damage (Baumann & Ganuldie,1994). These cytokines in tern stimulate neighboring cells to re-lease mediators that induce dialation of the local microvascularand cause permeabilization of capillaries. The accumulation ofcirculating leucocytes at the sites occurs by releasing chemo-tactic cytokines and lipid products as well as by expressingintercellular adhesion molecules. The invading leukocytes alsosynthesize mediators propagating the inflammatory response.Thus, there exists many events, specially the late one, for thedevelopment of edema that are not directly linked to the expres-sion of pro-inflammatory cytokines. Possibly for this reason,a prominent inflammatory change in the lungs was not visi-ble in spite of a high level of TNF-α and IL-6 in the BALFof 14- and 21-days GE-exposed mice. Alternatively, our studysuggests that the elevated expression of pro-inflammatory cy-tokine may not correlate with the inflammatory response at thesame time point and may be the reason for delayed effect. Incontrast, prominent thickening of alveolar septa, edema, influxof PMNs, and hypertrophied epithelial lining of bronchioles inlungs of rats chronically exposed to dilute diesel exhaust duringinflammatory response were observed (Henderson et al., 1988).Histopathological observation of GE-exposed mouse lung re-vealed a mild increase in lung-associated cell numbers, withoutconcurrent change in lavageable cell numbers, possibly becauseof the mild/gentle technique of lung lavage we adopted.

The increased activity of marker enzymes for lung dam-age, γ GT, ALP, and LDH in the BALF, further reinforces thepossibility of lung injury by GE. In the case of chronic expo-sure to dilute diesel exhaust, increase in the activities of LDHand ALP in lungs of rats has been reported (Henderson et al.,1988). Further, Sagai et al. (1996) reported chronic airways in-flammation characterized by the infiltration of eosinophils andlymphocytes, airway hyperresponsiveness, and mucous hyper-secretion following intratracheal instillation of diesel exhaustparticles in mice.

In this study, we assume IL-6 and TNF-α to play a piv-otal role in pulmonary inflammation following GE exposurethrough the inhalation route. However, we cannot exclude thepossibility that other mediators are also involved in expressionof lung injury in GE-exposed mice. Nonetheless, preventing theaccumulation of TNF-α in the BALF of silica-induced early fi-brogenic reaction in the lung by Nyctanthes arbortristis helpedto ameliorate lung injury (Paul et al., 2002). TNF-α has been

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proposed as an early and important mediator of acute lung injurybecause of its potent proinflammatory effects (Li et al., 1995).The release of TNF-α induces the production of IL-1β and otherproinflammatory cytokines, leading to the sequestration of de-granulation of neutrophils in the lungs (Moses et al., 1989).Therefore, agent(s) that block IL-6 and TNF-α may have ther-apeutic benefit for GE-induced pulmonary damage. Further-more, whether this increased production of proinflammatorymediators’ vulnerable effects balances in the immune system,resulting in an altered resistance to respiratory-tract infectionor altering the expression of respiratory allergy, is the subjectof further studies. Nonetheless, the study reveals that two ofthe proinflammatory cytokines (Il-6 and TNF-α) are elevated,and thus there is an indication of subtle change in the airwaysimmunobiology.

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