INTRODUCTION

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Ozone, one of the environmental oxidants, is known to decrease pulmonary function, increase airway hyperresponsiveness, and induce airway inflammation in humans (1), guinea pigs (6), and dogs (9). Many epidemiologic studies have suggested that asthmatics may be especially susceptible to the effects of ozone (10). It has been reported that the number of mast cells in bronchoalveolar lumen of asthmatics is significantly greater than that in normal subjects (13), and that the percentage of mast cells in bronchoalveolar lavage (BAL) cells is inversely and significantly correlated with the FEV₁, with the FEV₁%pred and with airway responsiveness to histamine (14). Moreover, the releasability of mast cells in the airways of asthmatics is greater than that of normal subjects (13, 14).

Histologically, mast cell degranulation (15) and migration (7) have been demonstrated in the lungs of some animals after ozone exposure, and further support is provided by the fact that exposure to ozone is associated with decreases in lung histamine in guinea pigs and mice (15). Moreover, nasal lavage fluid from normal human volunteers exposed to ozone contains elevated levels of tryptase, a mast cell granule enzyme (16). These results suggest that ozone may activate mast cells to release chemical mediators. Kleeberger and colleagues (17) demonstrated the important role of mast cells in acute ozone-induced lung inflammation by comparing the inflammatory responses in genetically mast-cell-deficient mice and mast-cell-sufficient congenic mice. However, the role of mast cells in ozone-induced airway hyperresponsiveness is still unclear.

In this study, we evaluated the importance of the mast cell in ozone-induced airway hyperresponsiveness in guinea pigs using tazanolast, an orally active mast-cell-stabilizing drug, which has been shown to suppress passive cutaneous anaphylaxis (18), Schultz-Dale reaction in isolated tracheal muscle, and experimental asthma without antagonistic actions upon histamine- and leukotriene-D4-induced contraction (19), IgE-mediated or compound 48/80-induced histamine release from mast cells and lung fragments (20, 21), compound 48/80-induced Ca²⁺ uptake into mast cells from extracellular medium (21), compound 48/80-induced translocation of protein kinase C from the cytosol to the membrane fraction of mast cells (21), and inositol trisphosphate production without directly inhibiting phospholipase C in mast cells (21).

	METHODS

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Animals

Male Hartley guinea pigs (Funabashi Farm, Shizuoka, Japan) weighing 400 to 600 g were used. The guinea pigs were kept in laminar flow isolation units of the animal research facility of Tohoku University School of Medicine. Animal care and handling were in accordance with the principles stated in the "Guide for the Care and Use of Laboratory Animals" (Prime Minister's Office of Japan, Publication No. 6, 1980). The experimental protocols described below were approved by the Animal Care and Use Committee of Tohoku University.

Drug

Tazanolast, butyl 3'-(1H-tetrazol-5-yl)oxanilate, was synthesized and supplied by Wakamoto Pharmaceutical Co., Ltd. (Kanagawa, Japan). Tazanolast was suspended in 0.5% carboxymethyl cellulose sodium solution for oral administration. Methacholine and carboxymethyl cellulose sodium were purchased from Sigma Chemical Co. (St. Louis, MO).

Ozone Exposure

Animals were put in a tightly sealed acrylic chamber (50 × 40 × 40 cm) with ducts to input and exhaust gas. Ozone was produced by a silent electric discharge generator (OT-26; Nippon Ozone, Tokyo, Japan), and was introduced with filtered carrier air at 25 L/min into the chamber. The concentration of ozone in the chamber was monitored by an ultraviolet ozone analyzer (Model 1003 AH; Dasibi, Glendale, CA) calibrated against a standard ozone source (Model 1410; Dylec, Ibaraki, Japan), and was maintained at 2.0 ± 0.2 ppm. Some animals were given sham exposure with carrier air alone.

Measurement of Respiratory Resistance

Respiratory resistance (Rrs) was measured by a method previously described (22). Briefly, an awake guinea pig was placed inside a body plethysmograph and a 30-Hz sine wave oscillation pressure generated by a 25-inch loudspeaker was applied to the body surface. The respiratory flow rate through a face mask and box pressure was measured. The 30-Hz component of the respiratory flow rate and box pressure with phase shift between these signals was detected by a two-phase lock-in amplifier (5610B; NF Electronic Instruments, Yokohama, Japan). Rrs was calculated using an analog computer.

Evaluation of Airway Responsiveness to Methacholine

Methacholine (MCh) aerosols were generated by an ultrasonic nebulizer (NE-U06; Omron, Kyoto, Japan) and were directed to a face mask placed on the guinea pig with a carrier flow of 10 ml/s. After baseline measurements were made, the guinea pigs were challenged with an aerosol of physiological saline and then methacholine aerosols. We defined Rrs after saline aerosol inhalation as the baseline Rrs. Animals were given MCh aerosols of 0.012, 0.025, 0.05, 0.1, 0.2, 0.4 and 0.8 mg/ml in physiological saline for 30 s at each concentration. Inhalation was repeated with aerosols of increasing concentrations at an interval of 1.5 min until the Rrs increased to more than twice the baseline value. Airway responsiveness to MCh was evaluated by the concentration of MCh required to increase the Rrs to twice the baseline value (PC200-MCh).

BAL

BAL was performed immediately after the last measurement of airway responsiveness. The guinea pigs were deeply anesthetized intraperitoneally with 50 mg/kg of pentobarbital sodium and were killed by exsanguination from the abdominal aorta. The trachea was cannulated with a polyethylene tube through which the lungs were lavaged three times with 10 ml of physiological saline (30 ml total). The BAL fluid was centrifuged at 150 × g for 10 min. The obtained pellet was immediately suspended in 4 ml of physiological saline, and total cell numbers in the BAL fluid were counted in duplicate with a hemocytometer (improved Neubauer counting chamber). Then, a 100-µl aliquot was centrifuged in a cytocentrifuge (Model 2 Cytospin; Shandon Scientific Co., Pittsburgh, PA). Differential cell counts were made from centrifuged preparations stained with Wright-Giemsa, counting 500 or more cells in each animal at a magnification ×1,000 (oil immersion).

Protocol

Experiment 1 (time course of airway responsiveness). On Day 1, PC200-MCh of eight guinea pigs was determined (preexposure PC200-MCh). On Day 2, the animals were put in the exposure chamber described above and were exposed to 2 ppm ozone for 2 h. At 1, 2, 4, and 6 h after ozone exposure, PC200-MCh was determined for each guinea pig.

Experiment 2 (time course of BAL). BAL was performed in five guinea pigs without ozone exposure. Then, the other 15 guinea pigs were exposed to 2 ppm ozone for 2 h, followed by BAL at 2, 4, and 6 h after the exposure for five animals.

Experiment 3 (effect of tazanolast on PC200-MCh after ozone exposure). On Day 1, preexposure PC200-MCh was determined for 50 guinea pigs. On Day 2, guinea pigs in groups of 10 were orally administered tazanolast at a dose of 30 mg/kg (T-30 group), 100 mg/kg (T-100 group), or 300 mg/kg (T-300 group), or vehicle alone (vehicle group). After 30 min, the animals were exposed to 2 ppm ozone for 2 h. In addition, the other 10 guinea pigs were also exposed to 2 ppm ozone for 2 h and then were orally administered 300 mg/kg of tazanolast (T-300 post group). At 2 h after exposure PC200-MCh of each animal was determined. Thereafter, BAL was performed for each animal.

Experiment 4 (sham exposure). On Day 1, the baseline PC200-MCh of 12 guinea pigs was determined. On Day 2, six guinea pigs were orally administered 300 mg/kg of tazanolast (T-300 sham group), and the others were given vehicle alone (sham exposure group). After 30 min they were given sham exposure with filtered air alone, and at 2 h after the exposure the PC200-MCh of each animal was determined. Thereafter, BAL was performed for each animal.

Statistical Analysis

The PC200-MCh and cell number in the BALF were analyzed using Wilcoxon's rank test. The decrease of PC200-MCh caused by ozone exposure was calculated with logarithmic values of preexposure and postexposure PC200-MCh, and was defined as "delta logPC200." All results are expressed as the mean ± SEM. A p value less than 0.05 was considered significant.

	RESULTS

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Airway Responsiveness

The correlation of PC200-MCh on Day 1 and that on Day 2 (after sham exposure) in the sham exposure group and the T-300 sham group is shown in Figure 1. The PC200-MCh did not vary significantly from day to day, and tazanolast per se at a dose of 300 mg/kg did not alter PC200-MCh.

View larger version (19K): [in this window] [in a new window]   Figure 1.   Correlation of PC200-MCh on Day 1 and that on Day 2 (after sham exposure) in the sham exposure group, n = 6 (open circles) and the T-300 sham group, n = 6 (closed circles). Solid line represents line of identity; dashed lines indicate 100% changes in PC200-MCh.

A representative time course of the airway responsiveness to methacholine after ozone exposure in a guinea pig of the vehicle group is shown in Figure 2. The time course of delta logPC200-MCh until 6 h after ozone exposure is shown in Figure 3. Airway responsiveness significantly increased from 1 h after exposure (p < 0.05), became the highest at 2 h (p < 0.01), and then gradually returned to the control level. PC200-MCh at 4 or 6 h after exposure was not significantly different from the preexposure value (p = 0.08 and p = 0.26, respectively). Because of these results we evaluated airway responsiveness at 2 h after exposure in the remainder of this study.

View larger version (20K): [in this window] [in a new window]   Figure 2.   A representative time course of the airway responsiveness to methacholine after ozone exposure in a guinea pig of the vehicle group. Closed circles, open circles, closed triangles, open triangles, and closed squares represent preexposure, 1, 2, 4, and 6 h after ozone exposure, respectively.

View larger version (41K): [in this window] [in a new window]   Figure 3.   Time course of delta log PC200-MCh after ozone exposure (2 ppm, 2 h) in guinea pigs (n = 8). Values with asterisks are different from the preozone values of that group (*p < 0.05, **p < 0.01).

The effect of tazanolast on the increase in airway responsiveness after ozone exposure is shown in Figure 4. The delta logPC200-MCh of the vehicle, T-30, T-100, and T-300 groups was 0.65 ± 0.07, 0.45 ± 0.05, 0.21 ± 0.08, and 0.09 ± 0.11, respectively. The values of T-30, T-100, and T-300 were significantly smaller than that of the vehicle group (p < 0.05, p < 0.01, and p < 0.001, respectively). However, the delta logPC200-MCh of T-300 post was not significantly different from that of the vehicle group. Thus, the increase in delta logPC200-MCh caused by ozone exposure was inhibited by tazanolast in a dose-dependent manner when it was administered before the exposure.

View larger version (34K): [in this window] [in a new window]   Figure 4.   Effect of tazanolast on ozone-induced airway hyperresponsiveness to inhaled methacholine in guinea pigs. Closed columns, open columns, shaded columns, hatched columns, and crosshatched columns represent vehicle, T-30, T-100, T-300 and T-300 post groups (n = 10 each), respectively. *p < 0.05, **p < 0.01 compared with the vehicle group.

BAL

The time course of BAL is shown in Figure 5. The number of neutrophils in BALF before and at 2, 4, and 6 h after the exposure was 2.3 ± 1.4, 12.8 ± 4.5, 18.5 ± 6.3, and 23.8 ± 8.7%, respectively; the number of neutrophils at 2, 4, and 6 h after the exposure was significantly greater than that before ozone exposure (p < 0.05 at any time point). Thus, the number of neutrophils continued to increase until 6 h after the exposure. In contrast, the number of macrophages, lymphocytes, or eosinophils did not change significantly after ozone exposure.

View larger version (30K): [in this window] [in a new window]   Figure 5.   Histograms of cells in BAL fluid recovered at different time points after ozone exposure. Closed columns, open columns, shaded columns, and hatched columns represent the results of BAL before and 2, 4, and 6 h after ozone exposure (n = 5 at all time points), respectively. **p < 0.01 compared with the group before ozone exposure.

The results of BAL at 2 h after ozone exposure are shown in Figure 6. The numbers of neutrophils in the vehicle, T-30, T-100, T-300, and T-300 post groups were significantly higher (p < 0.05 for all groups) than that in the sham exposure group, whereas there was no significant difference between any pair of groups except with the sham exposure group. There were no significant differences in the numbers of macrophages, lymphocytes, or eosinophils between any pair of groups. Tazanolast at any dose did not significantly change the cell distribution of BAL cells at 2 h after the exposure when compared with the vehicle group.

View larger version (31K): [in this window] [in a new window]   Figure 6.   Effect of tazanolast on cellular infiltration to the airway after ozone exposure in guinea pigs. BAL was performed at 2 h after ozone exposure. Striped columns, closed columns, open columns, shaded columns, hatched columns, and crosshatched columns represent the sham exposure, vehicle, T-30, T-100, T-300, and T-300 post groups (n = 10 each), respectively. The numbers of neutrophils in the T-30, T-100, T-300, and T-300 post groups were significantly higher (p < 0.05 for all the groups) than those in the sham exposure group, whereas there was no significant difference between any pair of groups except with the sham exposure group. There were no significant differences in the numbers of macrophages, lymphocytes, or eosinophils between any pair of groups.

	DISCUSSION

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Tazanolast does not affect the type II to type IV allergic reactions (systemic Forssman shock and reversed cutaneous anaphylaxis, passive Arthus reaction, contact dermatitis, and tuberculin reaction, respectively). Moreover, tazanolast has no effect on leukocytes, i.e., it does not inhibit histamine release from leukocytes of atopic patients stimulated with antigen, anti-IgE, or calcium ionophore A23187 (23) and nitroblue tetrazolium reduction test, phagocytosis, and chemotaxis in human neutrophils (24). Thus, tazanolast selectively inhibits mast cells to release histamine mainly by preventing the increase in the intracellular Ca²⁺ concentration (21). The reason why we adopted tazanolast in this study was that it can be taken orally and it does not possess a histamine receptor antagonizing action, which would modify the airway responsiveness to methacholine. We avoided administration of aerosolized drug to eliminate its influence on ozone inhalation.

In this study we demonstrated that tazanolast significantly inhibited ozone-induced airway hyperresponsiveness. First, we examined the time course of PC200-MCh after ozone exposure. Measurement of preexposure PC200-MCh was done on the day before ozone exposure in order to eliminate its influence on ozone inhalation. As shown in Figure 3, PC200-MCh began to decrease from 1 h after exposure, became the lowest at 2 h after exposure, and then gradually returned. Thus, we decided to evaluate airway responsiveness at 2 h after exposure in the remainder of this study. As shown in Figure 1, PC200-MCh did not change significantly after sham exposure in either group treated with vehicle alone or tazanolast at 300 mg/kg. These results indicated that day-to-day variation of PC200-MCh in each guinea pig was negligible in our exposure system and that tazanolast per se at a dose of 300 mg/kg did not alter PC200-MCh. The administration of tazanolast before ozone exposure inhibited ozone-induced airway hyperresponsiveness in a dose-dependent manner. However, administration of tazanolast after ozone exposure did not inhibit the increase of airway responsiveness. These results suggest that mast cells stimulated by ozone, and especially some chemical mediators released from mast cells, may play an important role in the development of ozone-induced airway hyperresponsiveness.

Several investigations have demonstrated that acute ozone exposure of humans (3, 5) and animal models (25, 26) in vivo elicits the release of mediators such as histamine, prostaglandins, LTB₄, peptidoleukotrienes, and thromboxane B₂. Lipoxygenase products derived from lung arachidonic acid metabolism have been suggested to be important in the pathogenesis of ozone-induced airway inflammation and airway hyperresponsiveness in guinea pigs (8). Although the cellular sources of these mediators during ozone exposure have not been determined, mast cells have the ability to release these mediators (27).

Some studies have demonstrated that neutrophilic infiltration into the airways causes ozone-induced airway hyperresponsiveness (2, 3, 9). However, Murlas and colleagues (7) demonstrated that the time course of the development of airway hyperresponsiveness preceded that of neutrophilic infiltration into the airways, which is consistent with our data. Moreover, it has been demonstrated that the depletion of neutrophils by cyclophosphamide or antibody fails to inhibit ozone-induced hyperresponsiveness in guinea pigs (28), mice (29), and dogs (30). These findings suggest that neutrophilic infiltration was a consequence of the airway damage rather than a cause of the development of ozone-induced airway hyperresponsiveness.

We conclude that tazanolast, a selective mast-cell-stabilizing drug, significantly inhibits ozone-induced airway hyperresponsiveness in guinea pigs. This result suggests that mast cell may play an important role in ozone-induced airway hyperresponsiveness.