INTRODUCTION

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Suplatast tosilate is a novel antiallergic agent that suppresses several processes, including the synthesis of IL-4 and IL-5 in both human and murine Th2 (-like) cells (1, 2); IgE synthesis in both mice and humans, with no direct effect on B cells (1, 3); local eosinophilia regulated by Th2 cells in mice (2); and infiltration of both eosinophils and CD4⁺ T cells into the airways, with consequent airway hyperresponsiveness after allergen inhalation in sensitized guinea pigs (4). Suplatast does not exhibit bronchodilating activity or antagonize spasmogens.

Antigen inhalation challenge in some allergic asthmatics induces an early asthmatic response (EAR) within 10 min (5), and some patients with an EAR subsequently develop a late asthmatic response (LAR) 3 to 8 h after challenge (6). The LAR, which may be considered to model a component of the inflammatory response in clinical asthma (7), is characterized by the infiltration of eosinophils (8). Airway responsiveness increases after the LAR, and the degree of the increase is correlated with the magnitude of the LAR (9). Patients with a LAR respond poorly to bronchodilators and often require corticosteroids for its resolution (6).

We have developed a guinea pig model of bronchial asthma in which both an EAR and a LAR, with abundant recruitment of eosinophils into the airways, follow antigen inhalation challenge (10, 11). In addition, increases in airway responsiveness appear after the LAR and last for 1 wk in this model (12). In the present study, we examined the inhibitory effect of suplatast on antigen-induced dual asthmatic responses and accumulation of inflammatory cells in airways in the guinea pig model. In addition, using in situ hybridization, we estimated expression of IL-5 mRNA in the lungs of this model after treatment with the high dose of suplatast or with vehicle only.

	METHODS

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Animals

Specific pathogen-free male Hartley guinea pigs (Charles River, Kanagawa, Japan) weighing 160 to 180 g were housed in 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

Suplatast tosilate (suplatast: (±)-[2-[4-(3-ethoxy-2-hydroxypropoxy) phenylcarbamoyl]ethyl]dimethylsulfonium p-toluenesulfonate) was synthesized and supplied by Taiho Pharmaceutical Co. Ltd. (Tokyo, Japan). Suplatast was dissolved in distilled water for oral administration.

Measurement of Pulmonary Resistance

The method of measuring pulmonary resistance (RL) has been described previously (11). RL was measured during spontaneous breathing. In brief, a guinea pig was anesthetized with an intraperitoneal injection of urethane (1.2 g/kg) and was laid quietly in a shaded cardboard box for 30 min until deeply anesthetized. After intracutaneous and subcutaneous injection of 4% xylocaine, an incision was made in the neck, the trachea was cannulated with an endotracheal tube, and the incision was sutured. A pneumotachograph coupled to a differential pressure transducer was connected to the endotracheal tube to measure airflow, and volume data were obtained by electrical integration of the flow signal with an integrator. Esophageal pressure was measured with a water-filled polyethylene tube and a pressure transducer. Airway opening pressure was measured with a pressure transducer connected between the endotracheal tube and the pneumotachograph. The transpulmonary pressure was determined by subtraction of airway opening pressure from esophageal pressure. The RL for each animal was calculated by the method of Mead and Whittenberger (13).

Experimental Protocol

Ascaris suum extract was prepared as previously described (11). Thirty guinea pigs were injected intraperitoneally with a mixture of 20 µg of A. suum protein and 20 mg of silica suspended in 1 ml of physiologic saline. The injection was repeated 2 wk later. One week after the second injection, the guinea pigs were given an aerosol of A. suum extract (2.5 mg/ml in physiologic saline) for 30 s through a face mask. The aerosol was generated with an ultrasonic nebulizer (NE-U06; Omron, Kyoto, Japan) and was administered at a constant flow rate of 10 ml/s. Beginning just before antigen inhalation and continuing once a day for 1 wk, groups of 10 animals were given suplatast at an oral dose of 30 mg/kg (S-30 group) or 100 mg/kg (S-100 group) or were given vehicle alone (control group). Blood samples for measurement of antigen-specific IgE and IgG were collected on Days 20 and 27 from five animals in the S-100 group and five animals in the control group. On Day 28, the operation described above was performed and the last dose of suplatast or vehicle was given. One hour later, the guinea pigs were challenged with an aerosol of physiologic saline and then with an aerosol of A. suum extract (2.5 mg/ml in physiologic saline). The aerosols (again generated with an ultrasonic nebulizer) were directed at a constant airflow (10 ml/s) into a cylindrical adaptor (volume = 10 ml) connected to the endotracheal tube. Each guinea pig inhaled the aerosol for 15 s. We defined baseline RL as the RL after saline aerosol inhalation. Subsequently, RL was measured at 5, 10, and 15 min and then on the hour until 6 h after inhalation. Airway secretions, if any, were removed with a paper string before each RL measurement. Immediately after the last RL measurement, the guinea pigs were deeply anesthetized with an additional dose of urethane and killed by exsanguination from the abdominal aorta. BAL was then performed and was followed by lung fixation.

To obtain a prechallenge group, we sensitized 10 additional guinea pigs in the same way until Day 27 and then performed BAL without the inhalation challenge on Day 28.

Antibody Measurements

Titers of antibody to A. suum were measured by homologous passive cutaneous anaphylaxis (PCA) reactions (14). Twofold dilutions of each serum sample were prepared in duplicate. One series of samples was used for the 1-wk PCA; the other was heated at 56° C for 4 h and used for the 4-h PCA. Samples were intradermally injected into the shaved backs of guinea pigs at a dose of 0.1 ml per site. A 1-ml volume of A. suum extract in physiologic saline (0.2 mg/ml) with 0.5% Evans blue was injected through a hind-foot vein 4 h after the intradermal injection in the 4-h PCA group and 1 wk later in the 1-wk PCA group. The highest dilution giving a 5-mm colored spot 30 min after injection was defined as the PCA titer. The 4-h PCA titer was taken to represent the IgG antibody titer, and the 1-wk PCA titer was considered to reflect the IgE antibody titer.

BAL and Lung Fixation

The right lung was lavaged twice through the endotracheal tube with 5 ml of physiologic saline. The lung was then inflation-fixed with periodate-lysine-paraformaldehyde via the trachea at 25 cm H₂O pressure; later, after being washed in 30% sucrose in phosphate-buffered saline, the lung was embedded in O.C.T. compound (Miles, Elkhart, IN). The BAL fluid thus obtained was centrifuged at 150 × g for 10 min. The pellet was immediately suspended in 5 ml of physiologic saline, and total cell numbers in BAL fluid were counted with a hemocytometer (improved Neubauer counting chamber). A 100-µl aliquot was then cytocentrifuged with Cytospin 2 (Shandon, Inc., Pittsburgh, PA). Differential cell counts were obtained from centrifuged preparations stained with Wright-Giemsa by counting  1,000 cells at a magnification of ×1,000 (oil immersion).

In Situ Hybridization

To prepare ribonucleotide probes for in situ hybridization, we cloned guinea pig IL-5 cDNA by the rapid amplification of cDNA ends method (15). The nucleotide sequence 48 to 541 was subcloned into pSPT18 (Boehringer Mannheim, Mannheim, Germany) and was used as a template for ribonucleotide probes.

Sections (5 µm) prepared from O.C.T.-embedded lungs were treated sequentially with 0.2 N HCl and pronase (25 µg/ml) at room temperature and were postfixed in 4% paraformaldehyde. The sections were then rinsed with phosphate-buffered saline, treated with glycine (2 mg/ml), and prehybridized for 60 min at 45° C in solution containing 50% deionized formamide and 2× SSC. Sections were then hybridized at 45° C in hybridization mixture (50% deionized formamide, 25 mM TRIS-Cl [pH 7.4], 2.5 mM EDTA, 1× Denhardt's solution, 0.3 M NaCl, 10% dextran sulfate, 1 mg of yeast tRNA/ml) containing 1 µg/ml of digoxigenin-labeled guinea pig IL-5 ribonucleotide probes (sense or antisense) prepared with a DIG RNA labeling and detection kit (Boehringer Mannheim). After 16 h, sections were washed in decreasing concentrations of SSC. Immunoreactive sites were blocked with proteolytic fragments of casein. The digoxigenin-labeled probe was detected with antidigoxigenin-alkaline phosphatase and visualized with nitroblue tetrazolium. For each section, three bronchovascular bundles were selected randomly, and IL-5 mRNA-positive cells, which were located around an arterial wall, including smooth muscle and a cuff of 100 µm outside the muscle, were counted with a video micrometer (VM-30; Olympus, Tokyo, Japan) in a blind fashion.

Statistical Analysis

The inhibitory effect of suplatast on RL was analyzed by Dunnett's test, and that on cell number in BAL was analyzed by the Steel test. Wilcoxon's test was used to compare IL-5 mRNA-positive cell counts in prechallenge versus control groups, in prechallenge versus suplatast groups, and in control versus suplatast groups. The PCA titers were statistically analyzed by Wilcoxon's test with use of logarithmic values for the concentrations. All results were expressed as the mean ± SE. A p value of < 0.05 was considered significant.

	RESULTS

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Time Course of RL

Because baseline values of RL for the control, S-30, and S-100 groups were similar (85.7 ± 11.3, 90.8 ± 16.5, and 84.0 ± 8.2 cm H₂O/L/s, respectively), changes in RL in these three groups after antigen inhalation challenge are expressed as percentages of each baseline value (Figure 1). In all guinea pigs, RL rapidly increased within 5 min and returned to near the baseline values during the next hour after antigen challenge. The maximal %RL values during the EAR in the S-30 and S-100 groups were not significantly different from that of the control group. Thereafter, however, the %RL of the S-100 group did not increase at all, whereas the %RL of the control group gradually increased again from 3 to 5 h after antigen inhalation, reflecting the LAR. The %RL of the S-30 group was significantly lower than that of the control group only 5 h after challenge. Thus, suplatast inhibited the appearance of the LAR in dose-dependent manner without inhibiting the EAR at all.

View larger version (14K): [in this window] [in a new window]   Figure 1.   Time course of pulmonary resistance (RL) after antigen inhalation challenge in the control group (open circles), n = 10, the S-30 group (closed triangles), n = 10, and the S-100 group (closed circles), n = 10. There was no significant difference in %RL between the three groups during the EAR. The %RL of the S-30 group was significantly lower than that of the control group only 5 h after challenge. The %RL of the S-100 group was significantly lower than that of the control group 3, 4, 5, and 6 h after challenge. *p < 0.05, **p < 0.02, ***p < 0.01, ****p < 0.005, and *****p < 0.00002 compared with the control group.

Antibody Titers

The IgG antibody titers on Day 20 in the control and S-100 groups were 2^{12.5 ± 0.3} and 2^{12.8 ± 0.2}, respectively, whereas the titers on Day 27 were 2^{16.5 ± 0.3} and 2^{16.6 ± 0.2}. The IgE antibody titers on Day 20 in the control and S-100 groups were 2^9.5 ± 0.3 and 2^9.8 ± 0.2, respectively, whereas those on Day 27 were 2^{12.8 ± 0.3} and 2^{13.0 ± 0.2}. Thus, there were no differences in IgG or IgE titers at either time point between these two groups.

BAL Analysis

There were no significant differences in recovery rates of BAL fluid for the control, S-30, S-100, and prechallenge groups (85.4 ± 1.9%, 84.5 ± 3.2%, 83.4 ± 2.3%, and 84.4 ± 3.1%, respectively). The numbers of cells in BAL fluid 6 h after antigen challenge of the control, S-30, and S-100 groups as well as in the absence of challenge (i.e., in the prechallenge group), are displayed in Figure 2. As would be expected, numbers of all cells in BAL fluid were significantly greater for the control group than for the prechallenge group. In addition, however, numbers of total cells, eosinophils, and lymphocytes in BAL fluid were significantly lower for the S-100 group than for the control group. Thus, suplatast specifically inhibited the recruitment of eosinophils and lymphocytes into the airways after challenge.

View larger version (19K): [in this window] [in a new window]   Figure 2.   Histograms showing distribution of cells in BAL fluid 6 h after antigen challenge in the control group (open columns), the S-30 group (closed columns), and the S-100 group (hatched columns), as well as in the absence of antigen challenge (i.e., the prechallenge group) (shaded columns). There were 10 animals per group. Numbers of all cells in BAL fluid were significantly greater for the control group than for the prechallenge group. Other significant differences are indicated by p values.

In Situ Hybridization

IL-5 mRNA-positive cells were found mainly between an artery and an adjacent bronchus (Figure 3). Because only a few positive cells were seen on the opposite side of the bronchus, we counted only the cells around arteries. The positive cells were classified as eosinophils and lymphocytes on the basis of distribution, shape of nucleus, and cell size in sections stained with Wright-Giemsa. As shown in Figure 4, antigen challenge significantly increased the number of positive cells after 6 h. One week of treatment with suplatast at a daily dose of 100 mg/kg statistically inhibited this increase, although the treatment did not inhibit IL-5 positive cells completely.

View larger version (150K): [in this window] [in a new window]   Figure 3.   In situ hybridization of lung tissue obtained 6 h after antigen challenge. (A) Distribution of IL-5 mRNA-positive cells in the lung of a guinea pig from the control group (original magnification: ×100). Positive cells were found mainly between an artery (a) and an adjacent bronchus (b). (B, C, and D) IL-5 mRNA expression around arteries in lungs from guinea pigs in the control group (B and C ) and the S-100 group (D) (original magnification: ×200; bar = 100 µm for all three panels). IL-5 sense probes were used for hybridization in panel C and antisense probes in the other panels.

View larger version (21K): [in this window] [in a new window]   Figure 4.   Number of cells (means ± SE) expressing IL-5 mRNA around arteries in the prechallenge group, the control group, and the S-100 group (seven animals per group). The number of positive cells in the S-100 group was significantly greater (p < 0.05) than that in the prechallenge group. Other significant differences are indicated by p values.

	DISCUSSION

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In all control guinea pigs, antigen inhalation challenge was followed by both an EAR and a LAR. During the LAR, eosinophils, lymphocytes, macrophages, and neutrophils accumulated in airways and numbers of IL-5 mRNA-positive cells increased in lung tissue. One week of treatment with suplatast at a daily dose of 100 mg/kg significantly inhibited the LAR without affecting the EAR. In addition, treatment specifically inhibited the recruitment of eosinophils and lymphocytes into airways 6 h after antigen challenge. Therefore, we concluded that suplatast inhibited the LAR by specifically inhibiting the inflammatory process after mast cell degranulation.

In our pilot study, a single 100-mg/kg dose of suplatast given before antigen challenge did not inhibit the LAR. Therefore, we used a 1-wk regimen in this study. Suplatast has been shown to inhibit IgE synthesis via inhibition of IL-4 synthesis but does not suppress the production of IgE by B cells already stimulated with IL-4 (3). We had previously found that antigen-specific IgE titers in the vehicle group increased within 3 wk after the first antigen injection. In this study, therefore, we administered suplatast from Day 21 through Day 28, i.e., after isotype switching to IgE in B cells had already occurred. As we intended, there was no significant difference between the control and S-100 groups in terms of specific IgE or IgG antibody titers on Day 28. This result is consistent with the failure of suplatast to inhibit the EAR significantly in this study protocol, although it is not yet clear why rather long-term administration is necessary for inhibition of the LAR.

Even though suplatast inhibited the LAR completely, there were still many eosinophils in BAL fluid. Therefore, we performed BAL in a prechallenge group and found that many eosinophils were already present in the airways before antigen challenge. These eosinophils were probably recruited as a result of antigen inhalation on Day 21. Because the prechallenge group had no asthmatic response, the presence of eosinophils is probably not sufficient to promote a LAR. Although we could not determine whether the failure was quantitative or qualitative, it is reasonable to conclude that the additional accumulation of eosinophils and lymphocytes in airways after challenge induced a LAR and that 1 wk of treatment with suplatast at a daily dose of 100 mg/kg inhibited the additional recruitment completely.

In atopic asthmatics, eosinophil infiltration into the airways is associated with activated CD4⁺ T cells (16). In BAL fluid from atopic asthmatics, CD4⁺ T cells have been found to express mRNA for IL-3, IL-4, IL-5, and GM-CSF, but not that for IFN- (17). In addition, airway eosinophilia in atopic asthmatics is known to be mediated by Th2 cytokines (18). Mansour and colleagues (21) had previously cloned guinea pig IL-5 cDNA; we also cloned the cDNA to examine whether suplatast inhibited the expression of the cytokine in this model. Through hybridization, we found that the expression of IL-5 mRNA in bronchovascular bundles significantly increased after antigen inhalation and that this increase was significantly inhibited by suplatast. Although some investigators (22) have reported that blocking of IL-5 inhibits antigen-induced eosinophil recruitment, late airway responses, and airway hyperresponsiveness in guinea pigs, we could not determine whether this mechanism was responsible for the inhibitory effects of suplatast on the LAR and on cell accumulation since the treatment did not inhibit IL-5-positive cells in lung tissues completely. In addition, since there is no monoclonal antibody to guinea pig IL-5, IL-5 mRNA-positive cells were classified as eosinophils and lymphocytes on the basis of distribution, shape of nucleus, and cell size in sections stained with Wright-Giemsa.

In conclusion, suplatast attenuated the LAR without affecting the EAR, inhibited eosinophil and lymphocyte infiltration into the airways, and decreased the expression of IL-5 mRNA in lung tissue to some degree. These results suggest that suplatast may be useful for the treatment of asthma.