Abstract
Diesel exhaust (DE) is a major air pollutant in urban areas. To clarify the effects of DE on the exacerbation of asthma, guinea pigs were exposed 12 h daily to 3 mg/m3 DE or air for 8 wk with or without sensitization to ovalbumin (OVA). In the DE-exposed sensitized animals, both immediate (IAR) and late (LAR) airway responses were enhanced after the inhalation challenge by OVA, compared with the DE-unexposed sensitized animals. Mucus was greatly accumulated in the airways of DE-exposed sensitized animals during IAR. The number of eosinophils and level of sialic acid concentration in bronchial lavage fluids were also significantly higher in the DE-exposed sensitized animals than in the DE-unexposed control animals. During LAR, intercellular spaces of the bronchial epithelium became enlarged in the DE-exposed sensitized animals, showing infiltration by numerous eosinophils. Albumin concentration was significantly higher in the bronchial lavage fluids from the DE-exposed sensitized animals than in those from the DE-unexposed control animals. These results suggest that exposure to DE enhances mucus hypersecretion and eosinophilic inflammation during IAR. DE exposure also increases airway permeability and airway inflammation during LAR. Thus, DE exposure exacerbates allergen-induced airway responses in guinea pigs.
Keywords: diesel exhaust; asthma; mucus; inflammation; guinea pigs
Asthma is a chronic respiratory disease that is increasing in both prevalence and mortality in developed countries, particularly in urban areas. Occupational and environmental exposures to sensitizers and irritants are causes of both asthma cases and exacerbation of asthma (1). Epidemiologic studies have shown that elevated levels of respirable particulate matter are associated with an increase in hospital admissions for asthma (2), and the particulate matters less than 2.5 μm (PM2.5) have a greater effect on the respiratory health of asthmatic children than those less than 10 μm (PM10) (3).
Diesel exhaust particles (DEP), produced mainly by diesel- engine–powered automobiles, are major participants in PM2.5. There are several findings indicating the relationship between DEP and allergic responses. The intranasal inoculation of DEP with the antigen enhances the antigen-specific IgE response (4). Intratracheal instillation of DEP also enhances allergen-related eosinophilic airway inflammation, airway hyperresponsiveness, and local expression of interleukin-5 (IL-5) and granulocyte macrophage-colony–stimulating factor (GM-CSF) in mice (5). Moreover, exposure to diesel exhaust (DE) enhances allergen-related eosinophil recruitment to the airways and increased protein levels of GM-CSF and IL-5 in the lungs (6). Although these results strongly suggest the interaction between DE(P) and allergic diseases, it is still unclear whether or not DE(P) enhances asthmatic responses in vivo.
We recently established an allergen-induced airway response model in guinea pigs that shows two-phase airway responses (immediate and late airway responses) resembling human asthmatic responses after antigen challenge (7). In this study, using this model, we investigated the effects of DE exposure on allergen-induced airway responses.
The experiments involved the use of female Hartley guinea pigs that were 4 wk of age at the initiation of the exposure (SLC farm, Shizuoka, Japan). They were housed in an animal facility that was maintained at 24 to 26 °C with 55% to 75% humidity and a 14-h/10-h light and dark cycle, and they were fed ad libitum. The studies adhered to the National Institutes of Health guidelines for the experimental use of animals. All animal studies were approved by the Institutional Review Board.
The guinea pigs were divided into four groups. The control group was exposed to filtered air without sensitization to ovalbumin (OVA) for 56 d. The OVA group was exposed to filtered air for 56 d. Seven days after the initiation of exposure, they were sensitized to 1 mg of OVA (Sigma Chemical, St. Louis, MO) emulsified in Al(OH)3 (100 mg in 1 ml sterile saline) by intraperitoneal injection. Three weeks after the primary sensitization, 10 μg OVA emulsified in Al(OH)3 was injected intraperitoneally as a booster. The DE group received 12 h of daily exposure to 3 mg/m3 of DE for 56 d, but they did not receive the sensitization to OVA. The DE+OVA group received the combined treatment following the same protocol as that for the OVA and the DE group. Fifty-six days after the initiation of exposures, the guinea pigs were challenged with inhalations of OVA (2 mg OVA per ml saline solution).
The guinea pigs were placed in a body-plethysmograph chamber equipped with a mouth-nose mask. The method for measurement of specific airway conductance (SGaw; s−1 cm H2O−1) has been reported previously (7).
Fifteen minutes and 4 h after the challenge with OVA, the lungs of anesthetized guinea pigs were lavaged with an initial 10 ml of phosphate-buffered saline (PBS). After centrifugation, the supernatant from this first lavage was used for an analysis of albumin (8), sialic acid (9), and CC chemokine concentrations. The concentrations of the CC chemokine eotaxin (Biosource International, Camarillo, CA), RANTES (Endogen, Cambridge, MA), and monocyte chemoattractant protein-1 (MCP-1; Endogen) were also measured, using an enzyme-linked immunosorbent assay (ELISA). The recovered cells were added to the products of additional lavage with PBS containing 3 mM EDTA for a total volume of 60 ml. A differential cell count was performed by standard light microscopic techniques.
For histopathologic assessment at a light microscopic level, paraffin sections were stained with hematoxylin-eosin and alcian blue. The lung tissues were also evaluated by transmission and scanning electron microscopy according to routine procedures.
The method of morphometric analysis has been reported previously (10). Forty goblet cells were evaluated in each group. The length of cilia was evaluated by measuring the maximal length of cilia in each cell at the electron microscopic level. Forty ciliated epithelial cells were evaluated in each group.
Serum was obtained from each group 56 d after the initial exposure. OVA-specific IgG1 antibody was evaluated in each group by using ELISA (Morinaga, Yokohama, Japan). OVA-specific IgE antibody was measured by using passive cutaneous anaphylaxis (PCA) as described previously (11).
Data were expressed as means ± SEM. SGaw data were analyzed using repeated-measures analysis of variance (ANOVA) at each time point. When a significant result in ANOVA was obtained, a Bonferroni post-test was used as a multiple comparison test. All other data were analyzed by a standard one-way ANOVA in combination with Duncan's multiple comparison test. A level of p < 0.05 was accepted as statistically significant.
The baseline value of sGaw did not significantly differ among the groups. Airway responses that were recognized as decreases in sGaw were observed in OVA-sensitized animals (OVA and DE+OVA groups) after the inhalation challenge with OVA. Immediate airway response (IAR) was observed 15 min after the challenge in the OVA group, and the response was resolved 1 h after the challenge (Figure 1). Late airway response (LAR) occurred 4 h after the challenge in the OVA group (Figure 1). Both IAR and LAR were significantly enhanced in the DE+OVA group, compared with the OVA group (Figure 1). Moreover, sGaw extended to be lower after IAR, and it never returned to the baseline value (Figure 1). Neither IAR nor LAR was observed in the animals in the control and DE groups (data not shown).
Fig. 1. Effects of diesel exhaust on airway responses after the inhalation challenge with ovalbumin. All values are expressed as mean ± SEM (n = 8) in each group); *p < 0.05.
[More] [Minimize]To assess the mechanisms of enhanced IAR in the DE+OVA group, histologic evaluations were conducted in the lungs of each group 15 min after the challenge. In the control and DE groups, the lung morphology did not differ from that before challenge (Figure 2A and 2B). In the OVA group, airway mucus was barely detectable around the bronchial epithelial cell layer (Figure 2C). In the DE+OVA group, mucus was widely spread throughout the bronchial lumen and formed mucus plugs 15 min after the challenge (Figure 2D). Eosinophils had infiltrated into the air spaces as well as the peribronchial area. The proportion of cells staining alcian blue did not differ among the four groups. Epithelial desquamation was not observed in any group.
Fig. 2. Airway sections of air-exposed ovalbumin-nonsensitized (A), DE-exposed ovalbumin-nonsensitized (B), air-exposed ovalbumin-sensitized (C ), and DE-exposed ovalbumin-sensitized animals (D) 15 min after the inhalation challenge with ovalbumin. Original magnification: ×200.
[More] [Minimize]Bronchial epithelial cells were also observed at the electron microscopic level during IAR. Mucus was located on the surface of the epithelial cell layer in the airways of the DE+OVA group 15 min after the challenge with OVA (Figure 3D). On the other hand, in the DE+OVA group, the amount of secretory granules was greatly decreased in goblet cells at that time point, compared with that in other groups (Figure 3A–3D). Short cilia were observed in bronchial epithelial cells of the DE and DE+OVA groups but not in the control and OVA groups (Figure 3A–3D). Eosinophils were located at the basal site of the epithelial cell layer in OVA and DE+OVA groups.
Fig. 3. Airway sections of air-exposed ovalbumin-nonsensitized (A), DE-exposed ovalbumin-nonsensitized (B ), air-exposed ovalbumin-sensitized (C ), and DE-exposed ovalbumin-sensitized animals (D) 15 min after the inhalation challenge with ovalbumin at an electron microscopic level. Arrowheads indicate eosinophils. Original magnification: ×1,700.
[More] [Minimize]Short cilia or loss of cilia was confirmed by use of a scanning electron microscope in DE-exposed animals during IAR (Figure 4A and 4B). Short cilia were also observed in DE-exposed animals before the challenge with OVA (data not shown).
Fig. 4. Scanning electron micrograph of airway surface of air-exposed ovalbumin-sensitized (A), and DE-exposed ovalbumin-sensitized animals (B) 15 min after the inhalation challenge with ovalbumin. Original magnification: ×3,500.
[More] [Minimize]The length of cilia was then evaluated in 40 ciliated epithelial cells in each group. Maximal length of cilia was measured in each cell at the electron microscopic level. The length of cilia was 4.77 ± 0.11 μm in the control group, which did not differ from that in the OVA group (Table 1). The lengths of cilia were significantly shortened in the DE and DE+OVA groups compared with values in the control and OVA groups (Table 1).
| Group | Cilia (μm) | |
|---|---|---|
| Control | 4.77 ± 0.11 | |
| DE | 1.33 ± 0.05†,‡ | |
| OVA | 4.80 ± 0.10 | |
| DE+OVA | 1.29 ± 0.04†,‡ |
Mucus secretion in IAR was further assessed morphometrically at the electron microscopic level. The volume densities of goblet cells in the bronchial epithelial cell layer were not different between the four groups at any time points (data not shown). The volume densities of secretory granules in goblet cells were not significantly different between the control, DE, and OVA groups 15 min after the challenge (Figure 5). The volume density of secretory granules in goblet cells was significantly lower in the DE+OVA group than in the other groups (Figure 5).
Fig. 5. The volume density of secretory granules in goblet cells 15 min after the inhalation challenge with ovalbumin. Forty goblet cells were analyzed in each group. *Significantly different from all other groups, p < 0.05.
[More] [Minimize]Airway mucus secretion was then evaluated in IAR using the first-recovered BAL fluids. The concentration of sialic acid, a component of mucus, in the BAL fluid recovered at 15 min after the challenge in the control group was 0.16 ± 0.01 μg/ml (Figure 6A). BAL fluid sialic acid concentration of both DE and OVA groups was not significantly different from that of the control group. The concentration of sialic acid was significantly higher in the BAL fluid from the DE+OVA group (0.67 ± 0.1 μg/ml) than in the other groups (Figure 6A).
Fig. 6. The concentration of sialic acid (A), and the number of eosinophils (B) in bronchoalveolar lavage fluids 15 min after the inhalation challenge with ovalbumin. Data are expressed as the mean ± SEM (n = 8) in each group. *Significantly different from all other groups, p < 0.05.
[More] [Minimize]Eosinophilic airway inflammation was evaluated by BAL cell analysis in IAR. The number of eosinophils recovered by BAL was 2.9 ± 1.0 × 105 in the control group 15 min after the challenge (Figure 6B). The number for both DE and OVA groups was not different from the control value. The number of lavageable eosinophils was significantly higher in the DE+OVA group (17.1 ± 0.9 × 105) than in the other groups (Figure 6B).
To assess the role of CC chemokine in eosinophil recruitment during IAR, the concentrations of eotaxin, RANTES, and MCP-1 were then evaluated in BAL fluid 15 min after the challenge. Eotaxin could not be detected in BAL fluid from the control, DE, and OVA groups at that time (Figure 7A). A significant amount of eotaxin was detected in the BAL fluid from the DE+OVA group during IAR (Figure 7A). Although low amounts of RANTES were observed in the BAL fluid from each group at that time, the amount did not differ among groups (Figure 7B). MCP-1 could not be detected in BAL fluid from any group during IAR.
Fig. 7. The concentrations of CC chemokine eotaxin (A) and RANTES (B) in bronchoalveolar lavage fluids 15 min after the inhalation challenge with ovalbumin. Data are expressed as the mean ± SEM (n = 8) in each group.
[More] [Minimize]To assess the mechanisms of the exacerbation of LAR in the DE+OVA group, morphologic features of the airway in LAR were evaluated in each group at the electron microscopic level. No pathologic findings were seen in the bronchus of the control group 4 h after the challenge (Figure 8A). Short cilia were observed in the bronchial epithelial cells but no other pathologic findings were seen in the DE group (Figure 8B). Intercellular spaces of bronchial epithelial cells were slightly enlarged, and eosinophils had infiltrated the spaces in the OVA group during LAR (Figure 8C). The gap between bronchial epithelial cells was greatly enlarged in the DE+OVA group at that time (Figure 8D). Numerous eosinophils had infiltrated the gap as well as the luminal surface in the DE+OVA group (Figure 8D).
Fig. 8. Airway sections of air-exposed ovalbumin-nonsensitized (A), DE-exposed ovalbumin-nonsensitized (B), air-exposed ovalbumin-sensitized (C ), and DE-exposed ovalbumin-sensitized animals (D) 4 h after the inhalation challenge with ovalbumin at electron microscopic levels. Arrowheads indicate eosinophils. Original magnification: ×1,700.
[More] [Minimize]To assess the airway permeability in LAR, the albumin concentration in the first recovered BAL was measured in each group. BAL fluid albumin concentration was 155 ± 12 μg/ml in the control group 4 h after the challenge, which was not different from the BAL albumin concentration before the challenge. The albumin concentration was not significantly altered in the BAL fluid from the DE group when compared with that from the control group (Figure 9A). The BAL fluid albumin concentration was significantly higher in the OVA group (785 ± 220 μg/ml) than in the control and DE groups 4 h after the challenge (Figure 9A). The concentration of albumin in BAL fluid was further enhanced in the DE+OVA group at the same time, compared with that in the OVA group (1,700 ± 90 μg/ml) (Figure 9A).
Fig. 9. Albumin concentration (A) and the number of eosinophils (B) in bronchoalveolar lavage 4 h after the inhalation challenge with ovalbumin. Data are expressed as the mean ± SEM (n = 8) in each group. *Significantly different in comparison with corresponding nonsensitized groups, p < 0.05. # Significantly different in comparison with corresponding air-exposed groups, p < 0.05.
[More] [Minimize]To assess the airway inflammation in LAR, BAL-recovered cells were analyzed in each group; 3.1 ± 1.8 × 105 eosinophils were recovered by BAL 4 h after the challenge in the control group (Figure 9B). The number of eosinophils in the DE group was not significantly different from the control value at that time. The eosinophils recovered by BAL were significantly increased in the OVA group (22.8 ± 5.0 × 105), compared with the control group (Figure 9B). The number of lavageable eosinophils was further increased in the DE+OVA group 4 h after the challenge (38.2 ± 4.0 × 105) when compared with that of the OVA group (Figure 9B).
To assess the adjuvant effect of DE exposure on OVA sensitization, serum levels of OVA-specific IgG1 and IgE antibodies were measured 56 d after the initial exposure. Both OVA-specific IgG1 and IgE antibodies were elevated significantly in the OVA and DE+OVA groups compared with values in the control and DE groups (Table 2). However, the serum levels of both OVA-specific IgG1 and IgE did not differ between the OVA and DE+OVA groups (Table 2).
In the present study, we have demonstrated that exposure to DE exacerbates allergen-challenged airway responses in vivo. DE exposure enhanced both IAR and LAR after the challenge with OVA. To our knowledge, this is the first study to demonstrate the effects of DE on the exacerbation of asthmalike airway responses in animal models.
In IAR, histologic evaluation and the analysis of BAL fluid suggested that the exacerbation of IAR in the DE+OVA group was in part due to mucus hypersecretion and eosinophilic airway inflammation. This was surprising because IAR is recognized as a result of airway muscle constriction alone by humoral factors, and airway inflammation is not often observed.
The formation of mucus plugs in the airways of the DE+OVA group was consistent with the histopathology of the airways during asthma exacerbation and near-fatal asthma in human subjects (12, 13). The increase in sialic acid concentration in BAL fluids also suggested the occurrence of mucus hypersecretion during IAR in the DE+OVA group. Morphologic and morphometric findings suggested that mucus hypersecretion was due at least in part to the secretion from goblet cells. Many inducers of airway goblet cell secretion have been reported, including lipid mediators (14, 15), a cholinergic agonist (16), substance P (17), and cytokines (18-20). Among these inducers, interleukin-4 (IL-4) has been shown to play an important role in both goblet cell metaplasia and mucus hypersecretion (18, 19). Although the mechanisms of mucus hypersecretion by DE exposure have yet to be elucidated, the finding that exposure to DE(P) enhances the pulmonary expression of IL-4 (5) did not contradict the pathophysiology of the present response.
In the present study, short cilia or loss of cilia was observed in DE-exposed animals, suggesting that the impairment of mucociliary clearance as well as mucus hypersecretion contributed to the exacerbation of IAR in the DE+OVA group. Because short cilia were observed in both DE and DE+OVA groups, this may have been a nonspecific change caused by DE exposure. Short cilia or loss of cilia is a common finding as a result of long-term exposure to air pollutants such as ozone (21) and nitrogen dioxide (22), and impairs mucociliary clearance.
In the present study, sGaw values continued to be lower after IAR in the DE+OVA group, whereas the values returned to what they were before the challenge in the OVA group. In the DE+OVA group, mucus was still widely spread after IAR (data not shown). It is therefore likely that the decrease in sGaw values after IAR was due to airway obstruction by mucus.
Eosinophilic airway inflammation is also often seen during the exacerbation of asthma in human subjects (23). Eosinophils activated at the tissue sites can release preformed toxic cationic granule proteins, oxidative products, lipid mediators, and cytokines, thus causing airway inflammation (24, 25). It has been suggested that eosinophil chemotactic factors, especially CC chemokines, play an important role in eosinophil recruitment during allergic diseases. A kinetic study of cytokine production showed that the expression of eosinophil chemoattractants such as eotaxin and RANTES increased after allergen inhalation, coincident with the peak number of activated eosinophils (26). In the present study, interestingly, eotaxin could be detected in BAL fluid from the DE+OVA group in IAR but was not detected in the BAL fluid from any of the other groups. On the other hand, the amount of RANTES in BAL fluids did not differ among the four groups in IAR. Moreover, MCP-1 could not be detected in BAL fluid during IAR in any group. These results suggest the specific contribution of eotaxin to eosinophilic airway inflammation in the DE+OVA group during IAR. This is a novel finding because eosinophilic inflammation has been considered one of the characteristic findings in LAR (27).
The present study also suggests that DE-mediated exacerbation of LAR is due to the increase in eosinophilic airway inflammation and the enhancement of airway permeability. Although such findings were observed in the OVA group, they were markedly enhanced in the DE+OVA group. The electron microscopic findings suggested that the increase in airway permeability was probably due to the enlargement of intercellular spaces of bronchial epithelium during LAR. E-cadherin is a calcium-dependent adhesion molecule expressed on epithelial cells, especially in adherence junctions, and it mediates mainly homophilic cell–cell adhesion (28, 29). We recently demonstrated that the dislocation of E-cadherin in the airway epithelium causes the adherence junctions to open and thus increases the airway permeability in LAR after antigen challenge (30). We are now investigating whether DE exposure enhances the dislocation of E-cadherin during LAR.
In the present study, serum levels of OVA-specific IgG1 and IgE antibodies did not significantly differ between OVA and DE+OVA groups, suggesting that DE-mediated exacerbation of airway responses was not due to any adjuvant effect. It is likely that nonspecific inflammation is involved in the pathogenesis of DE-mediated exacerbation of airway responses in our model.
Although the concentration of DE used in the present study was higher than the environmental level, the results obtained should make us pay more attention to the health effects of DE.
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