* Institute of Toxicology, College of Medicine, National Taiwan University, 1 Jen-Ai Rd., Section 1, Taipei, Taiwan, ROC; and Department of Applied Toxicology, Taiwan Agricultural Chemicals and Toxic Substances Research Institute, Taichung, Taiwan, ROC
Received February 11, 2004; accepted March 26, 2004
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ABSTRACT |
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Key Words: airway hyperresponsiveness; airway inflammation; asthma; IgE; intratracheal instillation; motorcycle exhaust particles.
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INTRODUCTION |
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Asthma is a chronic inflammatory disease of the lung that has been increasing in prevalence, morbidity, and mortality over the last two decades, and is characterized by reversible bronchospasm, airway inflammation, and airway hyperresponsiveness (AHR) (NHLBI, 1995). According to clinical data and animal models of asthma, it is suggested that, in allergic asthma, the Th2 lymphocytes induce, via the production of cytokines, an inflammatory cascade comprised of eosinophil activation, IgE production, and mast cell activation, all of which in turn produce the necessary mediators causing airway hyperresponsiveness (AHR) (Chung and Barnes, 1999
; Wills-Karp, 1999
). Although numerous studies have shown that DEP can exert biological effects that lead to the development or exacerbation of chronic allergic airway disease, full identification of the active components and their mechanisms has not yet been achieved. Other environmental factors might also be involved in the proinflammatory and proallergic responses.
In Taiwan, motorcycles are widely used, with more than 17 million registered in 2003, making the density as high as 0.85 motorcycles registered per person in the total population (MSTC, 2003). The use of motorcycles, especially the two-stroke engine, was estimated to introduce about 16,000 tons of total suspended particles (TSP) and 15,000 tons of particulate matter with a diameter of 10 µm (PM10), per year in Taiwan (EPA, Taiwan, 1994
). As compared with other countries, the mean concentration of PM10 per year is considerably higher in Taiwan (64 µg/m3) compared to some other parts of the world (London, U.K., 14 µg/m3; Paris, France, 14 µg/m3) (EPA, Taiwan, 1996
). Recent studies have highlighted the fact that PM10 or less can exacerbate asthma as well as chronic obstructive pulmonary disease (Diaz-Sanchez et al., 1994
; Nel et al. 1998
; Peterson and Saxon, 1996
). The impact of motorcycle exhaust particles (MEP) to the environment and its biological effects is relatively unclear. MEP contains a carbon black core, which absorb >110 different organic compounds, including C1C20 chains of hydrocarbon compounds and polycyclic aromatic hydrocarbons (PAHs) (Ueng et al., 2000
). Studies have indicated that MEP is cytotoxic (Lee and Kang, 2002
), mutagenic (Zhou and Ye, 1997
), and genotoxic (Kuo et al. 1998
) in vitro. It was also shown that MEP could impair the function of an isolated rat aorta (Cheng and Kang, 1999
) and several metabolic enzymes in rat tissues (Ueng et al. 1998
). However, no study has been carried out to examine the effects of MEP on the respiratory system, in vivo.
For the first time, the effect of MEP in inflammation-related lung parameters in laboratory animals was evaluated. We found that MEP, when applied intratracheally, would induce airway inflammation and AHR.
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MATERIALS AND METHODS |
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Collection and extraction of motorcycle exhaust particles.
The MEP were collected on 0.5-µm quartz fiber filters (Advantec MFS, Inc., CA) from 50-cm3 Yamaha Cabin or Suzuki two-stroke engines using 95% octane unleaded gasoline under the same conditions. The sampling apparatus consisted of, in sequence, a 30-cm long by 2.2-cm diameter stainless dilution tube, a filter holder, and a vacuum pump. Engines were running at idle speed of 150 rpm on an empty load and the vacuum pump was set at a flow rate of 20 l/min to collect MEP for 1 h twice daily. The quartz filters with collected particles were kept from light and left to dry in a desiccator, and were extracted four times with methanol under sonication. Methanol was then removed by vacuum evaporator. The weight change of the residue was frequently measured, and the complete removal of solvent was achieved when the weight of the residue was constant. The final residue, the MEP, was collected and kept desiccated in darkness at 20°C. Thirty-three µg of MEP could be extracted from one liter (l) of Yamaha motorcycle exhaust and 17 µg MEP from 1 liter of Suzuki motorcycle exhaust. In order to exclude the possible aging or any possible changes that might occur to the chemicals during storage, which might affect the biological activity of MEP. We have frequently treated the mice with MEP that has been stored for different periods of time after the preparation, and we have found that the MEP after storage for 2 months still gave the same effect as freshly prepared MEP.
For fractionation of the chemical components in MEP, the quartz filters with collected particles were extracted three times with 100 ml of hexane under sonication. The suspension was filtered and the residue was treated with the same volume of benzene, followed successively by chloroform, ethyl acetate, and methanol. The extracted fractions were filtered through a 0.22 µm filter to remove the particles, and the solvents were then removed by vacuum evaporator. The extracted residues by different organic solvents were collected and kept desiccated at 20°C. The following components are normally recovered from each gram of MEP: 0.81 g of n-hexane extract component, 0.09 g benzene extract component, 0.01 g chloroform extract component, 0.01 g ethyl acetate extract component, and 0.01 g methanol extract component.
Study protocol.
On Days 0, 14, and 28, mice were first anesthetized with 0.75 mg of ketamine and 0.06 mg of combelen per mouse, by intramuscular injection, and then applied intratracheally instillated with 50 µl of phosphate-buffered saline (PBS, pH 7.2) containing 24 µg (1.2 mg/kg) or 240 µµg (12 mg/kg) MEP collected from the Yamaha motorcycle. On both Day one and Day 7 after the last intratracheal instillation, mice were studied for airway hyperresponsiveness, bronchoalveolar lavage, and cytokine levels in BALF, serum total antibodies, and lung histology. By using trypan blue as a marker, we found that even distribution of the particle instilled throughout the lung could be achieved by using the technique of Leong et al. (1998) (data not shown). For most of the experiment, the MEP collected from the Yamaha motorcycle was used unless otherwise specified. In one experiment, MEP collected from the Suzuki motorcycle was used for comparison and fractionation. In that experiment, we used both 500 µg (25 mg/kg) of MEP or 500 µg (25 mg/kg) of residues from organic solvent-extracted fractions. For the dosing regimen selected, 12 mg/kg of MEP, collected from the Yamaha motorcycle, was one-tenth of the minimum lethal dose from an acute toxicity study (unpublished result). From a comparison study, we have found that 25 mg/kg of MEP collected from the Suzuki engine was needed to induce a similar degree of airway inflammation as that of 12 mg/kg of MEP collected from the Yamaha engine.
Lung histology.
The lungs were inflated with, and immersed in, 10% neutral phosphate-buffered formalin. Sections were prepared and stained with hematoxylin/eosin (H&E) to quantitate the number of infiltrating inflammatory cells under microscopy.
Bronchoalveolar lavage.
After treatment, mice underwent bronchoalveolar lavage using 1.5 ml sterile Hank's balanced salt solution (HBSS), instilled bilaterally with a syringe. The lavage fluid was harvested by gentle aspiration (Miyabara et al., 1998). This procedure was repeated three times. The fractions of lavage fluid were pooled, cooled to 4°C, and centrifuged at 300 x g for 5 min. Total cell counts were determined on fresh fluid specimens with the use of a hemocytometer. Differential cell counts were assessed on cytological preparations. The slides were prepared with the use of a Cytospin (Thermo Electron Corporation, Pittsburgh, PA) and stained with Liu staining. A total of 300 cells were counted under microscopy. Aliquots of supernatants were stored at 70°C and analyzed for cytokines by ELISA.
Determination of bronchoalveolar lavage cytokine level.
The lavage supernatants were utilized for determination of cytokine levels, IL-4, IL-5, TNF-, and INF-
. Cytokine levels were assayed by the ELISA method, according to the procedure recommended by the manufacturer (R&D, Minneapolis, MN. for IL-4 [interleukin-4], TNF-
, and interferon-
(IFN-
) [MedSystems Diagnostics GmbH, Vienna, Austria]; England. for IL-5.
Measurement of airway responsiveness.
Airway responsiveness was measured in unrestrained animals by barometric whole-body plethysmography (from Buxco, Troy, NY). Briefly, mice were placed in the main chamber, and baseline readings were taken and averaged for 3 min. Aerosolized PBS or methacholine (MCh) in increasing concentration (3.125 to 25 mg/ml) was nebulized through an inlet of the main chamber for 3 min, and readings were taken and averaged for 3 min after each nebulization. Recordings of every 10 breaths are extrapolated to define the respiratory rate in breaths per min. Airway reactivity was expressed as an enhanced pause (Penh) and data were expressed as the ratio of PenhMCh values to PenhPBS from three independent experiments (Hamelmann et al., 1997).
Measurements of total serum IgE and IgG2a.
Total serum IgE (indicative of Th2 response) and IgG2a (of the Th1 response) were measured by sandwich ELISA as previously described (Wu et al., 2000). Predetermined concentrations of anti-IgE and anti-IgG2a antibodies (Pharmingen, San Diego, CA) were coated onto ELISA plates and incubated at 4°C overnight. Plates were washed with PBS containing 0.05% Tween 20, and the diluted serum (50-fold dilution for IgE and 100-fold dilution for IgG2a determination) was added to the plates and incubated at room temperature for 2 h. Biotin-conjugated anti-IgE or anti-IgG2a antibodies and alkaline phosphatase-conjugated avidin (Sigma, St. Louis, MI) were added subsequently. Enzyme activity was evaluated using p-nitrophenyl phosphate (Sigma St. Louis, MI) as the substrate and read using an enzyme-linked immunosorbent assay (ELISA) reader (MRX-TC; Dynex Technology, Chantilly, VA). Readings at 405 nm were converted to ng/ml by using values obtained from standard curves. Standard curves were obtained from a series dilution of known concentrations of purified mouse IgE and IgG2a (Pharmingen, San Diego, CA).
Statistical analysis.
All values refer to means ± SEM (standard error of the mean) of at least three separate experiments. Statistically significant difference among groups was analyzed with one-way ANOVA followed by Scheffe for post hoc comparison using SPSS software. The minimal level of significance was a p value of <0.05.
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RESULTS |
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MEPs were sequentially extracted with different organic solvents, and the effect of each fraction was investigated. As seen previously, the cell numbers of macrophages, neutrophils, and eosinophils were increased in BALF at 25 mg/kg MEPs-treated mice (Table 3). In all the organic solvent extracts tested, only the benzene-extract fraction caused an increase in the numbers of neutrophil and eosinophil in BALF to a similar degree as that of the MEPs-treated group. Although the numbers of total cell and macrophage in BALF of the benzene extract-treated group were slightly increased, these were not significant when compare to the saline group.
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DISCUSSION |
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Airway inflammation is characterized by an increase of inflammatory-cell infiltration in that region. For example, airway inflammation in asthma is characterized by an eosinophilic inflammation (Bochner et al. 1994). We have found that the number of cells in BALF, including macrophage, neutrophil, lymphocyte, and eosinophil, were all increased after intratracheal instillation with MEP, especially at the 12-mg/kg concentrations. The infiltration and change of cell numbers of eosinophils and neutrophils was most obvious in the present study. The eosinophil is one of the major sources of cysteinyl leukotrienes that causes contraction of airway smooth muscle leading to AHR (Kay, 2001
). Whether neutrophils have a direct role in the development of AHR remains controversial, although the presence of neutrophils has been documented in allergic asthma and animal models of AHR (Devalia et al., 1996
; Kay, 2001
; Takano et al., 1997
; Tomkinson et al., 2001
). In addition to eosinophils and neutrophils, infiltration of macrophages was also observed in the MEP group. The infiltration of macrophages could also release cysteinyl leukotrienes, which play an important role in allergic asthma and development of AHR (Kay, 2001
). It has been shown that both eosinophils and neutrophils infiltrate BALF after repeated intratracheal instillation of DEP in mice (Sagai et al. 1996
).
Particle overload in the lung has been shown to cause chronic pulmonary inflammation by an impairment of alveolar macrophage-mediated lung clearance, which eventually leads to accumulation of excessive lung burdens (Morrow, 1992; Oberdörster, 1995
). One of the effective doses used in this study was 0.13 mg/g lung (1.2 mg/kg dosage), which was lower than that used in another particle overload experiment (1 mg dust/g lung tissue of F344 rat) (Morrow, 1986
). It was also shown that mice are less sensitive to "particle overload" than rats (Muhle et al. 1990
). In addition, the benzene-extracted fraction, which should be free of particles, induced similar effects as MEP. Based on these facts, we believe, although it needs to be proven, that the effects induced by MEP might be independent of particle overload.
In this study, we have found that MEP treatment could induce AHR, as judged by the increase of Penh, which represents a parameter of AHR by a noninvasive method. Different stimuli, direct and indirect, have been shown to induce AHR (Pauwel et al. 1988; Sterk et al. 1993
). Direct stimuli cause AHR by direct action on the effector cells such as airway smooth muscle cells, bronchial vascular endothelial cells, and mucus-producing cells. Indirect stimuli cause AHR by an action on cells such as inflammatory and neuronal cells, which then interact with effector cells (Van Schoor et al. 2000
). In a preliminary study, we have found that direct administration of MEP did not affect the contractility of the isolated trachea (unpublished result). This implies that induction of AHR by MEP might be caused indirectly through the inflammatory cells. Inflammatory cells, especially eosinophils, could release a myriad of mediators that are potentially important in AHR, including the eosinophil-specific proteins, cytokines, and lipid mediators (Wills-Karp, 1999
). In addition to the inflammatory cells, proinflammatory cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) has been shown to play an important role in DEP-induced AHR in mice (Ohta et al., 1999
). The role of GM-CSF and other mediators in MEP-induced AHR is still awaiting further investigation.
IgE plays a major role in the pathogenesis of allergic inflammation in humans, both in the early and late phases of the mucosal response to allergens. In addition, IgE is shown to cause antigen-specific eosinophil degranulation (Kaneko et al., 1995) and mast cell activation (Oettgen et al., 1994
). In the present study, we have seen that the serum IgE level was increased in MEP-treated mice. In contrast to IgE, the IgG2a level was not changed in MEP-treated mice, suggesting that the effect on IgE might be specific. Previous studies on DEP have shown that it could enhance the allergic inflammation by enhancing IgE production through different mechanisms, including cytokine production (Nel et al., 1998
) or direct activation of B cells (Takenaka et al., 1995
). In contrast to DEP, an in vitro study with isolated splenocytes showed that MEP did not cause cell proliferation by 3H-thymidine incorporation assay or IgE production (data not shown). This implies that the cytokine production may play a role in the induction effect of IgE production seen with MEP.
In this study, we have also observed that the several cytokine levels were increased in the BALF of MEP-treated mice. IL-5 can influence the production, maturation, and activation of eosinophils and the local differentiation of tissue-infiltrating eosinophil precursors (Chung and Barnes, 1999). The Th2 type cytokine, IL-4, can evoke the switch of antibody isotypes from IgM to IgE, which further mediates the development of an allergic response (Hurst et al., 2001
). The increase of IL-4 expression in BALF of the MEP-treated mice seems to agree with the increase of IgE production. It is interesting to note, we also observed that the IFN-
, a Th1 type cytokine, increased production in BALF in the higher dose of MEP-treated mice, although it has shown that IFN-
can inhibit the synthesis of IgE and the differentiation of precursor cells to Th2 cells. However, some evidence from in vivo studies conflicts with this hypothesis. For example, the level of IFN-
is elevated in the serum of severe asthma patients (Corrigan et al., 1990
) in supernatants from cultures of unstimulated and stimulated cells in BALF (Cembrzynaska-Nowal et al., 1993
). In the murine model, Hansen and colleagues (1990)
have found that introduction of Th1 cells worsened the underlying airway inflammation. Therefore, that the Th2 hypothesis is an answer in the MEP-inducing pathogenesis of asthma-like reaction seems an oversimplification; other possible mechanisms might be also involved (Salvi et al., 2001
).
A previous study by Kawasaki et al. (2001) has shown that the benzene fraction of DEP was important for the DEP-induced IL-8 gene expression in human epithelial cells. They also believed that BaP, a carcinogenic PAH, is an important active and major component of the benzene fraction, since BaP also induced IL-8 gene expression. We have fractionated the MEP by using organic solvents including hexane, benzene, chloroform, ethyl acetate, and methanol. We found that the benzene fraction caused similar effects as seen with MEP, such as an increase in eosinophil and neutrophil infiltration, IL-4 and IgE production, and AHR. This suggested that the chemicals responsible for inducing the effects observed might be enriched in the benzene fraction. Although both DEP and MEP could induce airway inflammation and AHR in mice, different chemical compositions and quantity might exist between MEP and DEP. The exhaust emitted from two-stroke motorcycle engines contained more hydrocarbons, both in quantity and species, than the diesel fuel (Chan and Nien, 1995
; Jemma et al. 1995
). Studies also showed that although the chemical composition of MEP, in terms of PAH content, is similar to that of DEP, the percentages are different (Barfknecht et al., 1982
; Hiura et al., 1999
; Ueng et al., 2000
). The PAHs composition found in DEP was: phenanthrene 52%, fluorenes 15%, naphthalene 13%, pyrene 10%, and fluoranthene 10% (Barfknecht et al. 1982
), and the composition found in MEP was: naphthalene 68%, anthracene 5%, acenaphthylene 4%, acenaphthene 4%, pyrene 4%, fluorine, 3%, fluoranthene 2%, and others 10%) (Ueng et al, 2000
). Therefore, the constitution and quantity of chemicals in the benzene-extracted fraction of DEP might be different from MEP. In addition, it has never been shown that BaP could induce airway inflammation by itself. Therefore, an important question to be answered is whether PAHs or other components are responsible for the effect seen with MEP. It is interesting to note that none of the MEP-extracted fractions induced an increase in the numbers of macrophages in BALF, as was observed in MEP. This implies that the carbon-black core, but not the chemicals absorbed in MEP, might be important in the induction of macrophage infiltration in the lung.
In conclusion, we have presented evidence showing that the filter-trapped particulate emissions from the unleaded-gasoline-fueled, two-stroke motorcycle engine may induce proinflammatory and proallergic response profiles in the absence of exposure to an allergen.
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ACKNOWLEDGMENTS |
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NOTES |
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1 To whom correspondence should be addressed. Fax: 886-2-3140217
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