Effects of diesel exhaust on allergic airway inflammation in mice

Effects of diesel exhaust on allergic airway inflammation in mice Yuichi Miyabara, PhD, Takamichi Ichinose, PhD, Hirohisa Takano, MD, Heung-Bin Lim, PhD, and Masaru Sagai, PhD Ibaraki, Japan

Background: Eosinophilic infiltration and goblet cell hyperplasia were induced by the intratracheal instillation of diesel exhaust particles and ovalbumin in mice. However, it is unknown whether its results differ from the effects of the inhalation of diesel exhaust and allergen. Objectives: The purpose of this study was to compare the effects of diesel exhaust inhalation and intratracheal instillation of diesel exhaust particles in a murine asthma model. Methods: ICR mice were exposed to 3 mg soot per cubic meter of diesel exhaust for 6 weeks. After the first week, animals were sensitized by intraperitoneal injection of ovalbumin and aluminum hydroxide gel. After 5 weeks of diesel exhaust exposure, the mice were challenged with ovalbumin. The animals were killed 1, 2, 3, and 7 days after the challenge and investigated for airway inflammation, hyperplasia of goblet cells, airway hyperresponsiveness, local cytokine expression, and antigen-specific IgE and IgG1 production. Results: Exposure to diesel exhaust enhanced infiltration of eosinophils and neutrophils in murine airways even 1 day after the challenge. An increment of goblet cells under the bronchial epithelium was followed by the recruitment of inflammatory cells. Furthermore, exposure to diesel exhaust combined with ovalbumin sensitization enhanced respiratory resistance and expression of IL-5 in lung tissue and IgG1 production but not IgE. However, diesel exhaust alone did not induce pathologic changes in mice. Conclusions: Diesel exhaust enhanced allergic airway inflammation, hyperplasia of goblet cells, and airway hyperresponsiveness caused by ovalbumin sensitization. (J Allergy Clin Immunol 1998;102:805-12.) Key words: Diesel exhaust, eosinophil, goblet cell, airway hyperresponsiveness, IL-5

Allergic asthma is a chronic disease of airway obstruction, airway inflammation, and enhanced bronchial responsiveness. Recently, the number of patients with allergic asthma among schoolchildren has been increasing steadily.1 The atmosphere in urban areas is heavily polluted with nitrogen dioxide (NO2) and suspended particulate matters. 2,3 Therefore there have been numerous attempts to demonstrate the relation between NO2 and allergic bronchial asthma. How-

From the Research Team for Health Effects of Air Pollutants, National Institute for Environmental Studies, Ibaraki, Japan. Received for publication Dec 31, 1997; revised June 25, 1998; accepted for publication June 25, 1998. Reprint requests: Yuichi Miyabara, PhD, Research Team for Health Effects of Air Pollutants, National Institute for Environmental Studies (NIES), 16-2 Onogawa, Tsukuba, Ibaraki 305-0053, Japan. Copyright © 1998 by Mosby, Inc. 0091-6749/98 $5.00 + 0 1/1/92699

Abbreviations used BALF: Bronchoalveolar lavage fluid DE: Diesel exhaust DEP: Diesel exhaust particles PBST: PBS with Tween 20 PC150ACh: Provocative concentration of acetylcholine causing 50% increase in respiratory resistance Rrs: Respiratory resistance

ever, no experimental data have demonstrated evidence that NO2 can induce allergic asthma.4-6 Recently, we showed that the repeated intratracheal instillations of diesel exhaust particles (DEP), which is a major part of suspended particulate matters in urban areas, induced chronic airway inflammation with infiltration of eosinophils and lymphocytes, hypersecretion of mucus, and airway hyperresponsiveness in mice.7 We have also developed a murine model of allergic asthma by the intratracheal instillation of DEP and ovalbumin.8 In this model, DEP enhanced eosinophilic airway inflammation, hyperresponsiveness, ovalbumin-specific IgG1 production, and IL-5 expression. This evidence suggests that DEP may enhance allergic asthma in human beings caused by allergens, which is increasing in modern buildings with airtightness.9 The effects of intratracheal instillation of DEP suspension in mice may differ from those of daily inhalation of diesel exhaust (DE). DE fumes are usually inhaled from the nose, but DEP suspensions are not taken directly into the lung. For this reason, it is worthwhile to evaluate the effects of inhaled DE on airway inflammation and hyperresponsiveness. Although there are many experiments on the effects of intranasal and intratracheal instillation of DEP suspension on allergic rhinitis and asthma models, none of the experimental studies have ever tried to show the effects of DE inhalation on allergic airway inflammation and hyperresponsiveness. To estimate the effects of DE exposure on human health, this inhalation experiment may be more worthwhile than the previous intratracheal or intranasal instillation studies. To elucidate the effects of DE on the expression of allergic airway inflammation caused by ovalbumin sensitization, we exposed mice to DE and sensitized with ovalbumin, then investigated airway inflammation, hyperplasia of goblet cells, airway hyperresponsiveness, local cytokine expressions, and the production of antigen-specific IgE and IgG1. 805

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METHODS Materials Acetylcholine, diethyl ether, thimerosal, phenylmethane sulfonyl fluoride, and Tween 20 were purchased from Nacarai Tesque (Kyoto, Japan). Rat anti-mouse IgE horseradish peroxidase–conjugated streptavidin, BSA, EDTA, 4-methyl-umbelliferyl-β-galactoside, and ovalbumin (grade V) were obtained from Sigma Chemical Co, Ltd (St Louis, Mo). Biotinylated rabbit anti-mouse IgG1 and βD-galactosidase–conjugated streptavidin were purchased from Zymed Laboratories (San Francisco, Calif). Leupeptin and pepstatin were purchased from Peptide Institute (Osaka, Japan). PBS (pH 7.4) was obtained from Nissui Pharmaceutical Co, Ltd (Tokyo, Japan). Diff-Quik staining solution was purchased from International Reagents Co, Ltd (Kobe, Japan). Rat anti-mouse IgE monoclonal antibody was obtained from Yamasa Shoyu Co, Ltd (Chiba, Japan). Schiff’s reagent was purchased from Merck (Darmstadt, Germany). All other chemicals were of the highest grade available.

Animals Male ICR mice (6 weeks old) were obtained from Japan Clea Co (Tokyo, Japan). They were fed commercial stock diet CE2 (Japan Clea Co) and water ad libitum. The animals were housed in a chamber that was maintained at 24° to 26° C with 55% to 75% humidity and a 14 hours/10 hours light/dark cycle. The study adhered to the National Institutes of Health guidelines for the use of experimental animals.

Generation of DE A 4JB-1–type, light-duty (2740 mL), 4-cylinder diesel engine (Isuzu Automobile Co, Tokyo, Japan) was connected to an ECDY dynamometer (Meiden-sha, Tokyo, Japan). The engine was operated with the use of standard diesel fuel at a speed of 1500 rpm under a load of 10 torque (kg/m).10 DE fumes were diluted with clean air to a constant particle concentration (3 mg soot/m3). The concentrations of nitric oxide (NO), NO2, sulfur dioxide (SO2), and carbon dioxide (CO2) in the diluted DE were 19.0 ± 2.4, 4.08 ± 0.29, 1.26 ± 0.10 and 3100 ± 220 ppm, respectively. The amounts of phenanthrene, fluoranthene, and pyrene in DEP were 268, 60, and 40 µg/g, respectively.

Study protocol Two hundred forty ICR mice were divided randomly into 4 groups: normal control (Air-saline); DE-saline; Air-ovalbumin; and DE-ovalbumin. The animals were exposed to clean air or diluted DE in separate chambers (2.2 m3) for 12 hours per day (10 PM to 10 AM) for 5 to 6 weeks. After the first 7 days, 2 groups of mice (Air-ovalbumin and DE-ovalbumin) were sensitized by intraperitoneal injection with 1 mg of ovalbumin dissolved in 0.5 mL of an aluminum hydroxide gel (alum) suspensions (3 mg/mL) in saline solution. After an additional 4 weeks of exposure, these mice (Air-ovalbumin and DE-ovalbumin) were challenged with ovalbumin by exposure for 15 minutes to an aerosol of 1% ovalbumin in saline administered through an ultrasonic nebulizer (NE-U07; Omron Co, Tokyo, Japan) in a chamber of 2000 cm3 volume. The 2 groups of unsensitized mice (Air-saline and DE-saline) were injected with saline instead of ovalbumin-alum and were challenged with a saline aerosol. These animals were killed 1, 2, 3, and 7 days after the challenge with saline or ovalbumin.

Histologic evaluation of eosinophils and goblet cells in lungs After exsanguination, the lungs were removed and fixed by intratracheal instillation with 10% neutral phosphate-buffered formalin

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at a pressure of 20 cm H2O. After at least 72 hours, slices (4 to 5 mm) of left pulmonary lobe were embedded in paraffin. Sections (3 mm thick) were prepared and stained with Diff-Quik to evaluate the infiltrated eosinophils. Eosinophils under the bronchial epithelium in each sample were observed with a micrometer (AX80; Olympus) under oil immersion. All bronchi of the whole sections of the left lung were examined, and the intensity of inflammation was graded in blinded fashion: (-), none; (±), very slight; (+), slight; (++), moderate; (+++), and moderate to marked; (++++), marked. “Very slight” was defined as less than 10% of the airway with eosinophilic infiltration; “slight” as 20% to 30% of the airway; “moderate” as 40% to 50%; “moderate to marked” as 60% to 70%, and “marked” as more than 80% of the airway. To evaluate goblet cells, the sections were stained with periodic acid Schiff. Goblet cells stained red in the bronchial epithelium were shown by the micrometer. The results were expressed as the intensity of hyperplasia of goblet cells in the same manner.

Bronchoalveolar lavage Bronchoalveolar lavage was performed 1 day after ovalbumin challenge. After collection of blood from the animals, the tracheae were cannulated. The lungs were lavaged with 1.2 mL sterile saline at 37° C, instilled bilaterally with a syringe. The lavaged fluid was harvested by gentle aspiration. This procedure was repeated 3 times. For all treatment groups, an average of 90% of the instilled 3.6 mL was retrieved. The fluids from 3 lavages were combined, cooled to 4° C, and centrifuged at 300g for 10 minutes. Total cell counts were determined on fresh fluid specimens with the use of a hemocytometer. Differential cell counts were assessed on cytologic preparations. The slides were prepared with the use of a Cytospin (Tomy Seiko) and stained with Diff-Quik. A total of 300 cells was counted under oil immersion microscopy.

Measurement of airway responsiveness Twenty-four hours after ovalbumin challenge, respiratory resistance (Rrs) was measured as described previously,11 with a minor modification. Briefly, the mice were anesthetized with pentobarbital sodium (50 mg/kg intraperitoneally), and a tracheotomy was performed with an 18-gauge cannula. The animals then were mechanically ventilated with a rodent respirator (model 683; Harvard Apparatus, South Natick, Mass) in a plethysmograph box with a pneumotachometer (BUXCO Electronics, Inc, Sharon, Conn) at a constant tidal volume (0.4 mL) and a rate of 120 breaths/min. Spontaneous respiration was inhibited by pancuronium bromide (1 mg/kg injected intramuscularly). The endotracheal pressure, flow, Rrs, and dynamic lung compliance were recorded continuously on a 6-channel recorder (BUXCO Electronics, Inc). The 4-second average of Rrs and dynamic lung compliance was also recorded. An acetylcholine challenge was performed by inhalation of an acetylcholine solution (0.08 to 1.25 mg/mL) for 2 minutes. The solution was aerosolized with an ultrasonic nebulizer (NE-U07; Omron Co). The provocative concentration of acetylcholine causing 50% increase in Rrs (PC150) value was calculated.

Quantitation of cytokine levels in lung-tissue supernatants One day after ovalbumin challenge, murine lungs were removed after the exsanguination. They were quickly frozen in liquid nitrogen and stored at –80° C until the assay was performed. Each lung was homogenized in 10 mmol/L potassium phosphate buffer (pH 7.4) containing 0.1 mmol/L EDTA, 0.1 mmol/L phenylmethane sulfonyl fluoride, 1 µmol/L pepstatin, and 2 µmol/L leupeptin. The homogenate then was centrifuged at 105,000g for 1 hour at 4° C. The supernatant was stored at –80° C.

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ELISAs for IL-5, GM-CSF, IL-2, IFN-γ, IL-10, and TNF-α were performed with the use of matching antibody pairs (Endogen, Cambridge, Mass). The ELISA for IL-4 was performed with the use of matching antibody pairs (Amersham, Buckinghamshire, England) according to the manufacturer’s instructions. The second antibodies were conjugated to horseradish peroxidase. Subtractive readings at 550 nm and 450 nm were converted to picograms per milliliter by using values obtained from standard curves generated with varying concentrations of recombinant IL-5, IL-4, GM-CSF, IL-2, IFN-γ, IL-10, and TNF-α. The detection limits of the assays were less than 5 pg/mL, 5 pg/mL, 5 pg/mL, 3 pg/mL, 15 pg/mL, 12 pg/mL, and 10 pg/mL, respectively.

Blood retrieval and analysis The mice were anesthetized with diethyl ether. The chest and abdominal walls were opened, and blood was retrieved by cardiac puncture. Plasma was prepared and frozen at –80° C until the assay for ovalbumin-specific IgE and IgG1.

Ovalbumin-specific IgE determination The titer of ovalbumin-specific IgE antibody was measured by ELISA.12 In brief, microplate wells (Dynatech, Chantilly, Va) were coated with an anti-mouse IgE rat monoclonal antibody at 37° C for 3 hours and incubated at 37° C for 1 hour with PBS containing 1% BSA and 0.01% thimerosal. After washing with PBS containing 0.05% Tween 20 (PBST), diluted serum samples were added to the wells and incubated overnight at 4° C. After washing with PBST, biotinylated ovalbumin was added to each well and incubated for 1 hour at room temperature. After washing, wells were incubated for 1 hour at room temperature with β-D-galactosidase–conjugated streptavidin. After the final washing, wells were incubated with 4-methylumbelliferyl-β-galactoside as the enzyme substrate at 37° C for 2 hours. The enzyme reaction was stopped with 0.1 mol/L glycine-NaOH buffer solution (pH 10.3). The fluorescence intensity was read by a microplate reader (Fluoroskan Flow Laboratories, Costa Mesa, Calif). Each plate included a previously screened standard plasma that contained a high titer of anti-ovalbumin antibodies. For standardization, the one titer was defined as twice the fluorescence units of preimmune plasma (blank). Ovalbumin-specific IgE titer in the well of each plate was calculated by using a standard curve made by serial dilution of the standard plasma and dilution factor. The diluted plasma sample, which is lower than 0.1 titer, was expressed as negative. ELISA antibody titer was expressed as the highest plasma dilution giving the positive reaction.

Ovalbumin-specific IgG1 determination Ovalbumin-specific IgG1 was measured by ELISA with a solidphase antigen. In brief, microplate wells were coated with ovalbumin overnight at 4° C; the plate was incubated at room temperature for 1 hour with 1% BSA-PBS containing 0.01% thimerosal. After washing with PBST, diluted serum samples were added to the wells and incubated at room temperature for 1 hour. After one more washing with PBST, the plate was incubated at room temperature for 1 hour with biotinylated rabbit anti-mouse IgG1. After another washing, wells were incubated with horseradish peroxidase–conjugated streptavidin at room temperature for 1 hour. Wells were washed and incubated with o-phenylenediamine and H2O2 in the dark at room temperature for 30 minutes. The enzyme reaction was stopped with 4N H2SO4. Absorbance at 490 nm was read by a microplate reader (model 3550, Bio-Rad Laboratories, Hercules, Calif). Each plate included standard serum that contained a high titer of anti-ovalbumin IgG1 antibodies. For standardization, the one titer was defined as twice absorbance of preimmune plasma (blank). Ovalbumin-specific IgG1 titer in the well of each plate was calculated by using a stan-

FIG 1. Histologic changes of eosinophilic inflammation in airway after ovalbumin (OVA) challenge with or without diesel exhaust exposure (DE). Mice were exposed to clean air or diesel exhaust for 5 to 6 weeks with ovalbumin sensitization. Lungs were removed 1, 2, 3, and 7 days after ovalbumin challenge. Degree of eosinophilic infiltration into submucosal layer was estimated: (-), none; (±), very slight; (+), slight; (++), moderate; (+++), moderate to marked; (++++) marked. n = 8 for each group. Significant difference was evaluated by Mann-Whitney U test. *P < .05 compared with Air-saline group, †P < .05 compared with DE-saline group, ‡P < .05 compared with Air-OVA group, §P < .05, compared with 1 day after challenge, ¶P < .05 compared with 2 days after challenge, #P < .05 compared with 3 days after challenge group.

dard curve made by serial dilution of the standard plasma and dilution factor. The diluted plasma sample, which is lower than 1 titer, was expressed as negative. ELISA antibody titer was expressed as the highest plasma dilution giving the positive reaction.

Statistical analysis Data are reported as mean ± SEM. Differences in the score of eosinophilic inflammation and hyperplasia of goblet cells were determined with nonparametric analysis (Statview; Abacus Concepts, Inc, Berkeley, Calif). The Mann-Whitney U test was used to distinguish between pairs of groups or days. Differences in the numbers of infiltrated inflammatory cells, airway hyperresponsiveness, cytokine protein levels, and immunoglobulin titers among groups were determined with analysis of variance (Statview). The Student t test was used to distinguish between pairs of groups.

RESULTS Histologic changes in murine airways after ovalbumin challenge with DE exposure DE exposure alone (DE-saline) did not induce the infiltration of eosinophils in murine lungs. However, DE exposure enhanced eosinophilic infiltration caused by ovalbumin sensitization (Fig 1). The number of eosinophils increased immediately after ovalbumin challenge in the DE-ovalbumin group and peaked 3 days after the challenge. Although ovalbumin sensitization alone

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FIG 3. Cellular profile in bronchoalveolar fluid (BALF). Mice were exposed to clean air or diesel exhaust (DE) for 5 weeks with or without ovalbumin (OVA) sensitization. Bronchoalveolar lavage was conducted 24 hours after ovalbumin challenge. Total cell counts were determined on fresh BALF, and differential cell counts were assessed with Diff-Quik staining. Results are expressed as mean ± SEM of 9 mice per group. *P < .05 compared with Air-saline group, †P < .05 compared with DE-saline group, ‡P < .05 compared with Air-OVA group. (Student t test).

FIG 2. Histologic changes of hyperplasia of goblet cells in airway after ovalbumin (OVA) challenge with or without diesel exhaust (DE) exposure. Mice were exposed to clean air or diesel exhaust for 5 to 6 weeks with ovalbumin sensitization. Lungs were removed 1, 2, 3, and 7 days after ovalbumin challenge. Degree of goblet cell proliferation, which reflected airway cell injury, was estimated: (-), none; (±), very slight; (+), slight; (++), moderate; (+++), moderate to marked; (++++), marked. n = 8 for each group. Significant difference was evaluated by Mann-Whitney U test. *P < .05 compared with Air-saline group, †P < .05 compared with DEsaline group, ‡P < .05 compared with Air-OVA group, §P < .05 compared with 1 day after challenge, ¶P < .05 compared with 2 days after challenge, #P < .05 compared with 3 days after challenge group.

number of eosinophils in the DE-ovalbumin group was twice that in the Air-ovalbumin group (Fig 3) (P < .05). These findings were consistent with the intensity of eosinophilic infiltration in lung tissue. DE exposure markedly enhanced the infiltration of neutrophils (DEovalbumin) after ovalbumin sensitization. The neutrophil population was increased 10.5-fold and 18.2-fold in the DE-ovalbumin group as compared with the Air-ovalbumin and DE-saline groups, respectively. DE and/or ovalbumin administration did not affect the total cell population, which consisted mainly of macrophages.

Airway hyperresponsiveness (Air-ovalbumin) also induced eosinophilic infiltration, the intensity of the infiltration was significantly lower after 1 day than in the DE-ovalbumin group. Goblet cells were observed in the airways of the DEovalbumin group as early as 1 day after ovalbumin challenge but were not observed in the Air-ovalbumin group (Fig 2). The number of goblet cells was markedly increased in the DE-ovalbumin group 7 days after ovalbumin challenge. The proliferation of goblet cells was significantly greater in the DE-ovalbumin group than in the Air-ovalbumin group at 2 and 7 days after challenge. Only a few goblet cells were observed in the nonsensitized groups (Air-saline and DE-saline). Eosinophilic infiltration and goblet cell hyperplasia were observed in the DE-ovalbumin group as early as 1 day after ovalbumin challenge but were not observed in the Air-ovalbumin group.

Cell composition in bronchoalveolar lavage fluids Eosinophilic infiltration was not observed in the nonsensitized groups (Air-saline and DE-saline). The number of eosinophils in bronchoalveolar lavage fluid (BALF) was increased in the Air-ovalbumin group; the

Although baseline Rrs in the Air-ovalbumin group was increased, the Rrs in the DE-ovalbumin group was increased by acetylcholine inhalation as compared with the other 3 groups. A decrease in the PC150 represents an increase in Rrs to acetylcholine. The PC150 values were significantly lower in the DE-ovalbumin, DE-saline, and Air-ovalbumin groups than in the Air-saline group (Fig 4) (P < .05). The PC150 in the DE-ovalbumin group was 0.15 mg acetylcholine/mL, which was significantly lower than that in the other 3 groups (0.30 to 0.64 mg acetylcholine/mL, P < .05), indicating that combined DE and ovalbumin treatment enhanced airway hyperresponsiveness to a greater degree than treatment with DE or ovalbumin alone.

Local cytokine expression DE inhalation alone enhanced expression of IL-10 and TNF-α, inhibited expression of IL-4 and GM-CSF, and did not influence expression of IL-5, IL-2, and IFN-γ (Table I). Ovalbumin sensitization alone induced the production of IL-5, IL-4, IL-2, and IFN-γ but did not affect the production of GM-CSF, IL-10, and TNF-α. Combined DE inhalation and ovalbumin sensitization enhanced the expression of IL-5, IL-2, and TNF-α com-

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pared with the Air-saline group. Combined treatment with DE and ovalbumin markedly increased the expression of IL-5 even 1 day after ovalbumin challenge. IL-5 expression in the DE-ovalbumin group was 1.5 times higher than in the Air-ovalbumin group. IL-2 was significantly increased by ovalbumin sensitization, but there was no significant difference between the Air-ovalbumin and the DE-ovalbumin groups. TNF-α was significantly increased by DE inhalation, but the production was lower in the DE-ovalbumin group than in the DE-saline group.

Ovalbumin-specific IgE and IgG1 production after ovalbumin challenge with DE exposure The ovalbumin-specific IgE titer was elevated 3 to 7 days after ovalbumin challenge. However, the increment in IgE in the DE-ovalbumin group was similar to that in the Air-ovalbumin group despite a marked difference in eosinophilic infiltration between the Air-ovalbumin and DE-ovalbumin groups (Table II). Thus DE inhalation did not enhance IgE production caused by ovalbumin sensitization in ICR mice. The IgE titer in both the Air-saline and DE-saline groups was below the detection limit (data not shown). The ovalbumin-specific IgG1 titer was elevated markedly 1 day after ovalbumin challenge in both the Air-ovalbumin and DE-ovalbumin groups and was markedly increased 7 days after challenge (Table II). The IgG1 titer was 3.2 times higher in the DE-ovalbumin group than in the Air-ovalbumin group 7 days after challenge. Thus DE exposure had an adjuvant effect on the production of ovalbumin-specific IgG1 induced by ovalbumin challenge but not IgE. The IgG1 titer in both the Air-saline and the DE-saline groups was below the detection limit (data not shown).

Analysis of correlation coefficients among examined factors The amount of IL-5 in lung tissue was correlated with the number of eosinophils in BALF (r = 0.805, n = 18, P < .0001). The pathophysiologic scores for eosinophilic infiltration and goblet cell proliferation also showed a highly significant correlation (r = 0.802, n = 64, P < .0001). The correlation coefficients between IL-2 and IL5, and IL-2 and the number of eosinophils in BALF were 0.893 and 0.888, respectively (P < .0001, n = 36). Although the IL-10 levels were correlated with TNF-α levels (r = 0.793, P < .0001), they did not correlate with other factors. Finally, no correlations existed between the amount of the amounts of IL-4, GM-CSF, or IFN-γ and the other factors examined.

DISCUSSION DE inhalation enhanced airway inflammation in association with marked eosinophil and neutrophil infiltration and airway hyperresponsiveness caused by antigen sensitization. Hyperplasia of goblet cells in the bronchial epithelium was followed by the recruitment of eosinophils. DE markedly enhanced the expression of IL-5 in murine lungs and the production of ovalbuminspecific IgG1 caused by ovalbumin sensitization. How-

FIG 4. Airway responsiveness to acetylcholine (ACh). Provocative concentration of acetylcholine causing 50% increase in respiratory resistance (PC150) values. Mice were exposed to clean air or diesel exhaust (DE) for 5 weeks with or without ovalbumin (OVA) sensitization. Respiratory resistance (Rrs) to acetylcholine was measured 24 hours after ovalbumin challenge. Baseline Rrs of Air-saline, DE-saline, Air-OVA, and DE-OVA groups were 1.12, 1.09, 1.23, and 1.19 cm H2O/mL per second, respectively. We monitored provocative concentration of acetylcholine causing 50% increase in Rrs (PC150) values. Results are expressed as mean ± SEM of 8 mice per group. *P < .05 compared with Air-saline group, †P < .05 compared with DE-saline group, ‡P < .05 compared with Air-OVA group (Student t test).

ever, DE inhalation alone for 5 weeks did not induce airway inflammation and airway cell injury in mice. To our knowledge, this study is the first demonstration that DE inhalation accelerates the pathogenic features of allergic asthma in mice. Previous studies evaluating the effects of DE in animals have used a DEP suspension instead of DE inhalation. Although the intratracheal or intranasal administration of DEP is useful for estimating the effects of particulate alone, these methods may not reflect the effects of daily inhalation of DE. Therefore the inhalation method is more useful model for evaluating the effects of air pollutants in human beings. However, DEP concentration (3 mg/m3) in this study is higher than in an actual urban atmosphere (<0.1 mg/m3). For risk assessment, further study is needed to investigate the effects of a much lower concentration of DE on these allergic reactions. The effects of DE exposure with ovalbumin sensitization on airway responsiveness, infiltration of eosinophils and neutrophils, hyperplasia of goblet cells, IL-5 expression, and ovalbumin-specific IgG1 production in mice were similar to the effects of intratracheal instillation of DEP with allergen. It indicates that particulate matter, unlike the gaseous component of DE, enhances pathogenic changes in vivo. In our previous study, the increase in IgG1 production preceded the increase in IgE production after intratracheal instillation of DEP and ovalbumin.8 In the current experiment, the production of IgG1 caused by ovalbumin challenge also preceded IgE production. These suggest that IgG1 may play an important role on the physiologic changes that characterize allergic asthma. The intranasal instillation of DEP and antigen has been found to increase antigen-specific IgE antibody in murine and human sera.13,14 However, DE did not enhance IgE production caused by ovalbumin sensitization in the current study. The discrepancy in study results

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TABLE I. Protein levels of cytokines in murine lung tissue supernatants Local cytokine expressions (pg protein/lung tissue supernatants) Group

n

IL-5

IL-4

GM-CSF

IL-2

IFN-γ

IL-10

Air-saline DE-saline Air-OVA DE-OVA

9 9 9 9

32.57 ± 2.93‡ 30.07 ± 2.24‡ 145.28 ± 37.43*† 217.73 ± 35.02*†

353.2 ± 11.8† 309.4 ± 11.8*‡ 390.9 ± 15.2† 387.0 ± 13.1†

33.87 ± 1.42† 26.07 ± 1.31* 30.02 ± 1.63 35.25 ± 1.94†

0.53 ± 0.31‡ not detected‡ 38.94 ± 14.54*† 44.68 ± 6.65*†

158.1 ± 41.9 130.6 ± 64.3 192.9 ± 56.9 223.2 ± 39.2

285.3 ± 22.2 365.3 ± 31.9‡ 265.4 ± 12.2† 321.8 ± 17.1‡

TNF-α

5026 ± 184† 8129 ± 492*‡ 5601 ± 232† 6664 ± 440*†‡

DE, Diesel exhaust; OVA, ovalbumin. Mice were exposed to clean air or DE for 5 weeks with or without OVA sensitization. Lungs were removed and frozen 24 hours after OVA challenge. Protein levels in lung tissue supernatants were analyzed with ELISA. Results are expressed as mean ± SEM of 9 mice per group. Significant difference was evaluated by Student t test. *P < .05 compared with Air-saline group; †P < .05 compared with DE-saline group; ‡P < .05 compared with Air-OVA group.

TABLE II. Ovalbumin-specific immunoglobulin titers in murine plasma OVA-specific IgE antibody log (titer)

1 day 2 days 3 days 7 days

OVA-specific IgG1 antibody log (titer)

Air-OVA group

DE-OVA group

Difference

Air-OVA group

DE-OVA group

Difference

<0.100 <0.100 0.151 ± 0.102 0.512 ± 0.209*

<0.100 0.170 ± 0.087 0.107 ± 0.073 0.638 ± 0.212*

— — P = .733 P = .681

3.161 ± 0.414 3.380 ± 0.394 3.986 ± 0.422 4.835 ± 0.339*

3.571 ± 0.353 3.427 ± 0.217 3.665 ± 0.228 5.341 ± 0.340*

P = .464 P = .917 P = .514 P = .309

DE, Diesel exhaust; OVA, ovalbumin. Mice were exposed to clean air or DE for 5 to 6 weeks with OVA sensitization. Serum was prepared 1, 2, 3, and 7 days after OVA challenge. IgE and IgG1 levels in serum were analyzed with ELISA. Results are expressed as mean ± SEM of 8 mice per group. Significant difference was evaluated by Student t test. *P < .05 compared with 1 day after challenge.

may be related to differences in the route of administration, the murine strain,15 the ovalbumin sensitization protocol, and the amount of DEP intakes. Intranasal instillation strongly stimulates the nasal membranes, but intratracheal administration does not. Inhalation of DE may stimulate the nasal membranes mildly. The intensity of the stimulation may relate to IgE production. The amount of ovalbumin and/or alum used differed among studies. In the current study, to investigate the effects of DE on allergic reactions, single ovalbumin instillation and challenge were done without booster. The protocol might be incomplete for sensitization, and IgE was not produced. The amount of DEP inhaled in the current experiment was estimated to be approximately 0.4 mg/wk per mouse. If we assume that 50% of DEP remained in the murine lung (0.2 mg/wk per mouse), the amount of DEP inhaled in the current study was twice that administered in our previous intratracheal study (0.1 mg/wk per mouse).8 These differences in experimental conditions may reflect the differences of IgE production. The mechanisms underlying allergic asthma remain unknown. To our knowledge, at least 3 independent pathways are thought to contribute to the physiologic changes that characterize allergic asthma. First, that the pathway of IgE and mast cells plays a central role in airway hypersensitive reactions is popular; IgE-mediated reactions are followed by chronic inflammation, leading to increased airway responsiveness. However, alternative and/or additional pathways of hypersensitivity reactions have been proposed. Immediate hypersensitivity and airway hyperresponsiveness have been induced by the administration of ovalbumin-specific IgG1 and IgE in mice,16 and allergen-

specific IgA, IgE, and IgG1 antibodies have been found to contribute to antigen-specific eosinophilic degranulation.17-20 Therefore, the second pathway, which eosinophilic degranulation induced by both antigen-specific IgE and IgG1 induces in murine airways, is suggested. Murine mast cells are also activated by IgG1 through FcγRIII as well as IgE.21,22 The activation of both inflammatory cells by immunoglobulins may lead to airway hyperresponsiveness in mice. Furthermore, allergeninduced bronchial hyperreactivity and eosinophilic inflammation have been observed in IgE-deficient and mast cell–deficient mice,23-25 and ovalbumin sensitization can induce eosinophilic inflammation, lung damage, and airway hyperreactivity without inducing the production of IL-4 or allergen-specific immunoglobulins in mice.26 Therefore, lung damage and airway hyperreactivity may be induced by eosinophilic degranulation without immunoglobulin productions. The degranulation, that is, the direct activation of eosinophils by inflammatory mediators, especially IL-5 from TH2 cells and other cells, is suggested as the third pathway of allergic asthma. In vivo nasal antigen challenge with DEP enhances the expression of mRNAs of IL-2, -4, -5, -6, -10, and -13 and IFN-γ in human beings.27 In our previous study, the intratracheal administration of DEP with ovalbumin enhanced the protein levels of IL-2, -4, and -5 and GM-CSF in murine lungs.8 In the current study, the expressions of IL-2 and IL-5 were markedly enhanced by DE inhalation with ovalbumin challenge. These results also indicate that both intratracheal administration and inhalation of DEP with ovalbumin enhanced both TH1- and TH2-type cytokine expression, especially expression of IL-5. Fur-

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thermore, IL-5 enhances both IgE and IgG1 production from B cells in the presence of IL-4.28,29 Therefore, IL-5 is thought to be a key cytokine in allergic airway inflammation through both the second and third pathways. In the current study, both the proliferation of goblet cells and airway inflammation preceded the increase in ovalbumin-specific IgE after ovalbumin challenge. DE exposure enhanced the production of ovalbumin-specific IgG1 induced by ovalbumin challenge but not IgE. These facts suggest that the second pathway (activation of eosinophils and mast cells by immunoglobulins) is superior to the first pathway (activation of mast cell by IgE), and DE inhalation enhances the second pathway. The amount of IL-5 in lung tissue increased 1 day after ovalbumin challenge. The amount of IL-5 in lung tissue was correlated with the number of eosinophils in BALF. The pathophysiologic scores for eosinophilic infiltration and goblet cell proliferation also showed a highly significant correlation. These results support the hypothesis that IL5 is important in the accumulation and infiltration of eosinophils and that airway obstruction and hyperresponsiveness follow the degranulations of eosinophils. On the other hand, DE consists of gaseous and particulate matters. Particles contain n-hydrocarbons, polyaromatic hydrocarbons, and quinones. Either NO2 or SO2 alone induces airway hyperreactivity but not IgE production and airway inflammation. The polyaromatic hydrocarbon fraction of DEP and phenanthrene, its major compound, enhance immunoglobulin production from B cells in vitro.30,31 Carbon black, as the core of DEP, also has adjuvant activity on local lymph node response and systemic IgE production in mice.32 DEP enhances release of inflammatory mediators such as IL-8, GM-CSF, and sICAM-1 from human bronchial epithelial cells in vitro. It may relate to the epithelial cell damage by superoxide and hydroxy radicals generated from quinones in DEP.33 As a result of these effects of DE components, especially release of inflammatory cytokines and adhesion molecules, DE fumes may induce and/or enhance airway hyperresponsiveness, inflammation, and epithelial damage. In conclusion, we demonstrated that the effect of DE exposure was enhancement of the eosinophilic accumulation, epithelial damage, and airway hyperresponsiveness in mice. However, these changes may occur independently, and we could not show whether DE fumes act on the sensitization step or on antigen challenge step. Further studies are needed to examine the mechanism of these pathogenic changes induced by DE inhalation.

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