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In vivo airway eosinophil accumulation does not enhance antigen- or propranolol-induced bronchoconstriction in guinea pigs
Prostaglandins & other Lipid Mediators 58 (1999) 219 –230
In vivo airway eosinophil accumulation does not enhance antigen- or propranolol-induced bronchoconstriction in guinea pigs Yoshihisa Ishiuraa,*, Masaki Fujimuraa, Shigeharu Myoua, Tokunao Amemiyaa, Kouichi Nobataa, Qi Liua, Hideki Tachibanaa, Tamotsu Matsudaa a
The Third Department of Internal Medicine, Kanazawa University School of Medicine, 13-1 Takara-machi, Kanazawa 920-8641, Japan Received 30 March 1999; received in revised form 30 April 1999; accepted 20 June 1999
Abstract Background: Chronic airway eosinophil accumulation is characteristic of asthma. However, it remains unclear whether airway eosinophils enhance or reduce release of chemical mediators and/or action of the released mediators in the airways in vivo, because previous investigators have indicated that eosinophil-derived factors such as histaminase and arylsulfatase may alter the allergic reaction by metabolizing chemical mediators. Recently, we have developed a guinea pig model of propranolol-induced bronchoconstriction (PIB), which is mediated by lipid mediators such as thromboxane A2 (TxA2), cysteinyl leukotrienes (cLTs) and platelet activation factor (PAF). This study was conducted to explain the influence of airway eosinophil accumulation on antigen-induced bronchoconstriction and the following PIB, both of which are mediated by lipid mediators. Methods: Guinea pigs were transnasally treated with 75 g/kg of polymyxin-B or vehicle twice a week for a total of 3 weeks. Guinea pigs were anesthetized and treated with diphenhydramine hydrochloride, and then artificially ventilated 24 h after the last administration of polymyxin-B or vehicle followed by passive sensitization. Propranolol at a concentration of 10 mg/ml was inhaled 20 min after an aerosolized antigen challenge. Results: The proportion of eosinophils in bronchoalveolar lavage fluid obtained 15 min after the propranolol inhalation was significantly increased in guinea pigs treated with polymyxin-B compared with the vehicle. The polymyxin-B treatment did not affect antigen-induced bronchoconstriction or the following PIB.
* Corresponding author: Division of Pulmonary Medicine, Toyama Red Cross Hospital, 2-1-58 UshijimaHonmachi, Toyama City, Toyama 930-0859, Japan. Fax: ⫹81-764-33-2274. 0090-6980/99/$ – see front matter © 1999 Elsevier Science Inc. All rights reserved. PII: S 0 0 9 0 - 6 9 8 0 ( 9 9 ) 0 0 0 4 0 - 4
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Conclusions: We conclude that eosinophils accumulated in the airways by polymyxin-B does not affect release of chemical mediators induced by antigen or propranolol inhalation, or action of released mediators in vivo. © 1999 Elsevier Science Inc. All rights reserved. Keywords: Polymyxin-B; Airway eosinophil accumulation; Allergic bronchoconstriction; Propranolol-induced bronchoconstriction; Guinea pigs
1. Introduction Bronchial asthma is characterized by a reversible airflow limitation and bronchial hyperresponsiveness (BHR) to specific and nonspecific stimuli that is considered to depend on airway inflammatory cells, mainly eosinophils [1,2]. Several investigators have reported that the BHR to methacholine and histamine significantly correlates to the percentage of eosinophils in bronchoalveolar lavage (BAL) fluid [3–5], and that chemical mediators, such as thromboxane A2 (TxA2), cysteinyl leukotrienes (cLTs), and platelet-activating factor (PAF) enhance bronchial responsiveness [6 – 8]. However, recent investigators have failed to demonstrate the relationship between the BHR and the number of eosinophils in biopsied bronchial tissues [9 –13]. On the other hand, previous researchers have indicated that some eosinophil-derived factors may alter the allergic reaction via metabolizing chemical mediators released by allergic reaction; histaminase and arylsulfatase may limit or possibly terminate the allergic reaction [14 –20]. Moreover, we have reported that BHR to methacholine is not present in patients with bronchodilator-resistant cough with sputum eosinophilia and cough hypersensitivity (atopic cough) [21] and eosinophilic pneumonia [22], despite the increase of eosinophils in bronchial mucosa. Gibson et al. [23] have also shown that sputum eosinophilia is not associated with BHR. Therefore, the exact role of eosinophils in the airways in vivo remains unknown. Propranolol can cause bronchoconstriction only in asthmatic patients [24,25]. We recently have shown that propranolol inhalation induces bronchoconstriction within a few minutes after propranolol is inhaled 20 min after a challenge with aerosolized antigen in passively sensitized and artificially ventilated guinea pigs [26]. Although the precise etiology of propranolol-induced bronchoconstriction (PIB) remains unclear, some researchers have proposed that cholinergic nerve activity may play a role [27]. Mast cells in human lung possess -receptors on their surface [28]. -Adrenoreceptor agonists have been shown to inhibit the anaphylactic release of mediators, including histamine, cLTs (LTC4, LTD4, and LTE4) and TxA2 [29]. Histamine has been shown to be involved in PIB in asthmatic patients [30]. Our previous animal studies have demonstrated that TxA2, cLTs and PAF are important in the PIB [26,31,32]. These findings suggest that inflammatory mediators contribute to the development of PIB [26,31,32]. This study was designed to explore whether in vivo airway eosinophil accumulation enhances or reduces antigen-induced bronchoconstriction and/or the following PIB, both of which are mediated by lipid mediators [26,31,32].
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2. Materials and methods 2.1. A model of eosinophilic bronchitis Male, albino, Hartley strain guinea pigs weighing 200 g were obtained from Sankyou Laboratory Service (Toyama, Japan). They were transnasally treated with 75 g/kg of polymyxin-B twice per week for a total of 3 weeks. For each procedure, animals were mildly anaesthetized by diethyl ether. Control animals were administered normal saline as the vehicle in the same manner. 2.2. Passive sensitization of guinea pigs Guinea pig homocytotropic antiserum was made by the modified method of Santives et al. [33]. Briefly, 500 g of ovalbumin (OA) was emulsified in Freund’s complete adjuvant and injected intradermally into each guinea pig at multiple sites. A booster dose was prepared and administered in the same manner 2 weeks later. Serum was collected from each animal 2 weeks after the booster dose, pooled, and kept frozen until use. The antibody titer of this serum was 1:12 800, 1:6400, and 1:512, as estimated by passive cutaneous anaphylaxis at 4 h, 24 h, and 7 days, respectively. Normal guinea pigs were passively sensitized with 1 mL of antiserum/kg intraperitoneally immediately after the last administration of polymyxin-B or vehicle. 2.3. Installation of artificial respiration Guinea pigs were anesthetized by an intraperitoneal injection of 75 mg/kg sodium pentobarbital and placed in the supine position (Fig. 1) 24 h after the last administration of polymyxin-B or normal saline, followed by passive sensitization. The trachea was cannulated with a polyethylene tube (2.5 mm outside diameter and 2.1 mm inside diameter). The animals were artificially ventilated by using a small animal respirator (Model 1680, Harvard Apparatus Co., South Natick, MA, USA) adjusted to a tidal volume of 10 mL/kg at a rate of 60 strokes/min. The changes in lung resistance to inflation, the lateral pressure of the tracheal tube (pressure at the airway opening; Pao, cm of H2O), were measured by using a pressure transducer (Model TP-603T, Nihon Koden Kogyo Co., Ltd., Tokyo, Japan). Because we [34] demonstrated that the changes in the Pao after an antigen challenge in passively sensitized guinea pigs treated with and without antihistamines and after an inhalation of LTC4 represented the averages of the changes in pulmonary resistance and reciprocal dynamic lung compliance, we used change in the Pao as an overall index of the bronchial response to bronchoactive agents. When all procedures were completed, the animals were administered diphenhydramine hydrochloride (60 mg/kg intraperitoneally) to block the action of histamine. By clamping the outlet port of the respirator, we overinflated the animals by two times tidal volume for two breaths [34]. All the animal procedures in this study complied with the standard set out in the guidelines for the care and use of laboratory animals in Takara-machi campus of Kanazawa University.
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Fig. 1. Design of the experimental system. An anaesthetized guinea pig was placed in the supine position and the trachea was cannulated with polyethylene tube. The animal was artificially ventilated by a small animal respirator and nebulized with ovalbumin and propranolol. The Pao was continuously recorded to estimate the OA and the propranolol-induced bronchoconstriction by a X–Y recorder.
2.4. Experimental protocols 2.4.1. Study 1: Influence of airway eosinophil accumulation on antigen- and propranololinduced bronchoconstriction The guinea pigs were assigned into two groups (Fig. 2): control group and polymyxinB-treated group (n ⫽ 8 in each group). Fifteen minutes after the installation of artificial respiration, when the Pao had stabilized, the passively sensitized animals were challenged with nebulized OA dissolved in physiologic saline (1 mg/mL) without interrupting the constant ventilation. The OA aerosol was generated for 30 s by using an ultrasonic nebulizer developed for small animals at our institution [35]. The amount of aerosol was 15.2 L/min, and 46.4% of the aerosol was deposited in the lung as measured by the radio-aerosol technique [35]. Twenty minutes after the OA provocation, 10 mg/mL of propranolol was inhaled for 30 s. 2.4.2. Study 2: Influence of single transnasal administration of polymyxin-B on bronchial responsiveness to inhaled methacholine The guinea pigs were assigned into two groups: control group and polymyxin-B treated group (n ⫽ 8 in each group). Polymyxin-B in a dose of 75 g/kg or saline as the vehicle was transnasally administered 24 h before this experiment. Fifteen minutes after the installation of artificial respiration, when the Pao had stabilized, ascending doses of methacholine (12.5, 25, 50, and 100 g/mL) were inhaled for 20 s through the nebulizer at intervals of 5 min. 2.5. Analysis of BAL cells Cell composition of BAL fluid was conducted 15 min after the inhalation of propranolol in Study 1, and 5 min after the inhalation of 100 g/mL of methacholine in Study 2. Each
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Fig. 2. Experimental protocols. Group 1, passively sensitized guinea pigs treated with repeated intranasal administration of saline; group 2, passively sensitized guinea pigs treated with repeated intranasal administration of polymyxin-B; group 3, nonsensitized guinea pigs treated with single transnasal administration of saline; group 4, nonsensitized guinea pigs treated with single transnasal administration of polymyxin-B.
BAL fluid was compared between animals treated with polymyxin-B and saline. Ten milliliters of sterile saline were instilled, at room temperature, through a polyethylene cannula introduced into the trachea, and recovered immediately. Following this manner, the same procedure was repeated; a total of 20 mL of sterile saline was used for BAL. After filtrating the fluid through a double layer of Dacron nets, cells were centrifuged at 1000 rev./min for 12 min and resuspended in 3 mL of RPMI-1640 (Grand Island Biological Co., Grand Island, NY, USA). Following dilution with an equal volume of Tu¨rk solution, the number of cells was counted in a Bu¨ker chamber. The fluid was then resuspended with RPMI-1640 to make a cell suspension containing 2 ⫻ 105 cells/mL. Smears for differential cell counts were prepared by cytocentrifugation (Cytopin 2, Shandon Southern Products Ltd., Cheshier, UK) at 800 rev./min for 8 min. After staining with May-Gru¨nwald-Giemsa stain (Diff-Quick, American Scientific Products, McGraw Park, IL, USA), 300 cells were counted. 2.6. Chemicals The following chemicals were used: sodium pentobarbital (Abbot Laboratories, North Chicago, IL, USA), polymyxin-B sulfate (Pfizer Taito Co. Ltd., Tokyo), diethyl ether (Wako Pure Chemical IN, Osaka, Japan), OA (Sigma, St. Louis, MO, USA), diphenhydramine hydrochloride (Sigma), and dl-propranolol hydrochloride (Wako Pure Chemical Ind.)
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Fig. 3. BAL cell findings. BAL was performed 15 min after an inhalation of propranolol following an OA challenging in passively sensitized guinea pigs treated with repeated intranasal administration of saline (open bars, n ⫽ 8) or polymyxin-B (shaded bars, n ⫽ 8). Error bars indicate mean ⫾ SEM. *p ⬍ 0.05 and **p ⬍ 0.01 compared with the saline group.
2.7. Statistical analysis One-way ANOVA was used for analyzing difference in baseline Pao value or each cell component in BAL fluid between groups. The degree of bronchoconstriction was evaluated using a time course curve for the percentage increase in the Pao from the preantigen challenge value induced by antigen provocation or propranolol inhalation, and was analyzed by repeated measure ANOVA. These analyses were performed using the software StatView 4.02 (Abacus Concepts, Berkeley, CA, USA). A p value of less than 0.05 was considered statistically significant, and values were expressed as the mean ⫾ SEM.
3. Results 3.1. Study 1: Influence of airway eosinophil accumulation on antigen- and propranololinduced bronchoconstriction The total number of effector cells recovered per milliliter in BAL fluid was significantly different ( p ⬍ 0.05) between treatment with polymyxin-B (8.8 ⫾ 2.9 ⫻ 105 cells/mL) and vehicle (saline) (5.3 ⫾ 0.7 ⫻ 105 cells/mL). Bronchoalveolar cell findings are shown in Fig. 3. The proportion of eosinophils in BAL fluid was significantly increased in guinea pigs treated with polymyxin-B compared with the saline. There were no significant differences between the groups about the proportion of neutrophils and lymphocytes in BAL fluid. Baseline Pao value before OA challenge did not differ significantly between animals treated with polymyxin-B (10.2 ⫾ 0.6 cm of H2O) and saline (10 ⫾ 1.3 cm of H2O). Fig. 4 shows time course of the percentage increases in the Pao after OA provocation followed by propranolol inhalation. The time-course curve for the percentage increases in the Pao after the OA challenge or the following propranolol inhalation did not differ between the two groups.
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Fig. 4. Time course of percentage increases in Pao after an aerosolized OA challenge followed by an inhalation of propranolol in passively sensitized guinea pigs treated with repeated intranasal administration of saline (open circle, n ⫽ 8) or 75 g/kg polymyxin-B (closed circle, n ⫽ 8). Vertical bars represent SEM. NS ⫽ not significant.
3.2. Study 2: Influence of single transnasal administration of polymyxin-B on bronchial responsiveness to inhaled methacholine The total number of effector cells recovered per milliliter in BAL fluid was not significantly different between treatment with polymyxin-B (13.8 ⫾ 1.5 ⫻ 105 cells/mL) and vehicle (saline) (10.1 ⫾ 1.9 ⫻ 105 cells/mL). Bronchoalveolar cell findings are shown in Fig. 5. There was no significant difference in the proportion of neutrophils, eosinophils or lymphocytes in BAL fluid between the groups. Fig. 6 shows the percentage of increases in the Pao caused by inhalation of ascending doses of methacholine. Baseline Pao value before methacholine inhalation did not differ significantly between animals treated with polymyxicin B (9.4 ⫾ 0.3 cm of H2O) and saline (9.2 ⫾ 0.2 cm of H2O). Percentage increases in the Pao produced by each dose of methacholine did not differ between the two groups.
Fig. 5. BAL cell findings. BAL was performed 5 min after final inhalation of methacholine in nonsensitized guinea pigs treated with repeated intranasal administration of saline (open bars, n ⫽ 8) or polymyxin-B (shaded bars, n ⫽ 8).
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Fig. 6. Percentage of increases in Pao after inhalation of each dose of methacholine in nonsensitized guinea pigs treated with repeated intranasal administration of vehicle (open circle, n ⫽ 8) or 75 g/kg polymyxin-B (closed circle, n ⫽ 8). Vertical bars represent SEM. NS ⫽ not significant.
4. Discussion In this study, airway eosinophil accumulation induced by repeated transnasal administration of polymyxin-B did not alter antigen-induced bronchoconstriction or subsequent PIB in passively sensitized guinea pigs pretreated with diphenhydramine in vivo. Recently, chronic desquamative eosinophilic bronchitis has been considered to be a fundamental feature of bronchial asthma [36,37] and significant correlation between the degree of BHR and airway eosinophil accumulation in stable asthmatics has been demonstrated [3–5]. The role of eosinophil-derived mediators in the development of BHR has also been postulated because of the significant correlation between the concentration of major basic protein and the number of eosinophils in BAL fluid in asthmatics [3]. However, a recent study indicates that eosinophil-derived major basic protein inhibits antigen-induced BHR in sensitized guinea pigs [38]. Sanjar and colleagues [11,12] also have reported that there is no significant increase in the airway reactivity after PAF challenge or antigen challenge by aerosol in guinea pigs, despite the airway eosinophil accumulation. Sasaki et al. [39] have shown that interleukin-5, which is known as a chemotactic factor for eosinophils, does not increase bronchial responsiveness in guinea pigs, and others have failed to demonstrate the relationship between the BHR and the number of eosinophils in biopsied bronchial mucosa in asthmatics [9,10,13]. Moreover, we have shown that bronchial responsiveness to methacholine is not increased in patients with bronchodilator-resistant cough with sputum eosinophilia and cough hypersensitivity (atopic cough) [21] and eosinophilic pneumonia [22], despite the increase of eosinophils in biopsied bronchial mucosa. Gibson et al. [23] have also shown that sputum eosinophilia is not associated with BHR. Although previous investigators demonstrated the generation of cLTs and PAF by eosinophils in vitro [40 – 42], others indicated that some eosinophil-derived factors may alter the allergic reaction by metabolizing or inactivating chemical mediators: histaminase and arylsulfatase may limit or possibly terminate the allergic reaction [14 –20]. These findings described above suggest that eosinophilic inflammation may not be
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essential for the development of bronchial asthma and BHR. The exact role of eosinophils in bronchoconstriction mediated by intrinsic mediators in vivo also remains unclear. Thus, we conducted this study to explain the influence of airway eosinophil accumulation on antigen-induced bronchoconstriction and PIB in passively sensitized guinea pigs in vivo. Polymyxin-B used in this study is a mast cell degenerating agent [43], and is known to promote the accumulation and activation of eosinophils [44]. We recently developed an animal model of eosinophilic bronchitis induced by polymyxin-B and have demonstrated the increase in the number of accumulated eosinophils in the mucosa and the injury to airway epithelium in trachea, large and medium bronchi, but not in the alveolar region or the lung structure [45]. The degree of activation of eosinophils recruited in this study has remained unknown, because we did not measure it. However, we consider that eosinophils recruited by polymyxin-B are activated in vivo as Davis et al. [46] have shown it. It is well known that most of the known mediators are involved in allergic bronchoconstriction [47], and our previous animal studies have also demonstrated the involvement of lipid mediators in pathogenesis of PIB [26,31,32]. Although this animal model of PIB can not evaluate dose response relation to propranolol, it can assess the effect of propranolol in a time-course study. As the mediator mechanism appears to play an important role in both antigen-induced bronchoconstriction and PIB, our animal model for PIB after antigeninduced bronchoconstriction is considered able to evaluate the influence of various factors on bronchoconstriction mediated by chemical mediators, except for histamine. In the present study, a 3-week transnasal administration of polymyxin-B did not alter antigen-induced bronchoconstriction or after PIB, despite the increased number of airway eosinophils as proven in BAL fluid. We also showed that a single transnasal administration of polymyxin-B did not affect bronchial responsiveness to inhaled methacholine. As our previous study regarding airway eosinophil accumulation revealed that repeated transnasal administration of polymyxin-B does not affect bronchial responsiveness to inhaled histamine despite increased infiltration of eosinophils in intrapulmonary small airways [45], we can evaluate the influence of airway eosinophil accumulation on bronchoconstrictor response mediated by intrinsic lipid mediators in this animal model. Although additional remarks may be done for the sensitization and challenge with OA and the association of polymyxin B plus antigen challenge, another previous study indicated that antigen challenge does not cause allergic airway reaction in nonsensitized animals [26]. As mentioned previously, our results imply that eosinophils accumulated by repeated transnasal administration of polymyxin-B in vivo do not potentiate or prevent chemical mediator release such as TxA2, cLTs, and PAF, which has been proven to be involved in both the antigen-induced bronchoconstriction and the following PIB in our animal model [26,31, 32]. We conclude from this study that in vivo eosinophil accumulation in the airways does not influence releasability of mediators from effector cells or inactivation of released mediators. Further studies may be required to explore the influence of eosinophil accumulation for histamine releasability in allergic bronchoconstriction and the role of mast cells, neutrophils, and other cells in bronchoconstriction mediated by various intrinsic mediators in vivo.
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In vivo airway eosinophil accumulation does not enhance antigen- or propranolol-induced bronchoconstriction in guinea pigs Yoshihisa Ishiuraa,*, Masaki Fujimuraa, Shigeharu Myoua, Tokunao Amemiyaa, Kouichi Nobataa, Qi Liua, Hideki Tachibanaa, Tamotsu Matsudaa a
The Third Department of Internal Medicine, Kanazawa University School of Medicine, 13-1 Takara-machi, Kanazawa 920-8641, Japan Received 30 March 1999; received in revised form 30 April 1999; accepted 20 June 1999
Abstract Background: Chronic airway eosinophil accumulation is characteristic of asthma. However, it remains unclear whether airway eosinophils enhance or reduce release of chemical mediators and/or action of the released mediators in the airways in vivo, because previous investigators have indicated that eosinophil-derived factors such as histaminase and arylsulfatase may alter the allergic reaction by metabolizing chemical mediators. Recently, we have developed a guinea pig model of propranolol-induced bronchoconstriction (PIB), which is mediated by lipid mediators such as thromboxane A2 (TxA2), cysteinyl leukotrienes (cLTs) and platelet activation factor (PAF). This study was conducted to explain the influence of airway eosinophil accumulation on antigen-induced bronchoconstriction and the following PIB, both of which are mediated by lipid mediators. Methods: Guinea pigs were transnasally treated with 75 g/kg of polymyxin-B or vehicle twice a week for a total of 3 weeks. Guinea pigs were anesthetized and treated with diphenhydramine hydrochloride, and then artificially ventilated 24 h after the last administration of polymyxin-B or vehicle followed by passive sensitization. Propranolol at a concentration of 10 mg/ml was inhaled 20 min after an aerosolized antigen challenge. Results: The proportion of eosinophils in bronchoalveolar lavage fluid obtained 15 min after the propranolol inhalation was significantly increased in guinea pigs treated with polymyxin-B compared with the vehicle. The polymyxin-B treatment did not affect antigen-induced bronchoconstriction or the following PIB.
* Corresponding author: Division of Pulmonary Medicine, Toyama Red Cross Hospital, 2-1-58 UshijimaHonmachi, Toyama City, Toyama 930-0859, Japan. Fax: ⫹81-764-33-2274. 0090-6980/99/$ – see front matter © 1999 Elsevier Science Inc. All rights reserved. PII: S 0 0 9 0 - 6 9 8 0 ( 9 9 ) 0 0 0 4 0 - 4
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Conclusions: We conclude that eosinophils accumulated in the airways by polymyxin-B does not affect release of chemical mediators induced by antigen or propranolol inhalation, or action of released mediators in vivo. © 1999 Elsevier Science Inc. All rights reserved. Keywords: Polymyxin-B; Airway eosinophil accumulation; Allergic bronchoconstriction; Propranolol-induced bronchoconstriction; Guinea pigs
1. Introduction Bronchial asthma is characterized by a reversible airflow limitation and bronchial hyperresponsiveness (BHR) to specific and nonspecific stimuli that is considered to depend on airway inflammatory cells, mainly eosinophils [1,2]. Several investigators have reported that the BHR to methacholine and histamine significantly correlates to the percentage of eosinophils in bronchoalveolar lavage (BAL) fluid [3–5], and that chemical mediators, such as thromboxane A2 (TxA2), cysteinyl leukotrienes (cLTs), and platelet-activating factor (PAF) enhance bronchial responsiveness [6 – 8]. However, recent investigators have failed to demonstrate the relationship between the BHR and the number of eosinophils in biopsied bronchial tissues [9 –13]. On the other hand, previous researchers have indicated that some eosinophil-derived factors may alter the allergic reaction via metabolizing chemical mediators released by allergic reaction; histaminase and arylsulfatase may limit or possibly terminate the allergic reaction [14 –20]. Moreover, we have reported that BHR to methacholine is not present in patients with bronchodilator-resistant cough with sputum eosinophilia and cough hypersensitivity (atopic cough) [21] and eosinophilic pneumonia [22], despite the increase of eosinophils in bronchial mucosa. Gibson et al. [23] have also shown that sputum eosinophilia is not associated with BHR. Therefore, the exact role of eosinophils in the airways in vivo remains unknown. Propranolol can cause bronchoconstriction only in asthmatic patients [24,25]. We recently have shown that propranolol inhalation induces bronchoconstriction within a few minutes after propranolol is inhaled 20 min after a challenge with aerosolized antigen in passively sensitized and artificially ventilated guinea pigs [26]. Although the precise etiology of propranolol-induced bronchoconstriction (PIB) remains unclear, some researchers have proposed that cholinergic nerve activity may play a role [27]. Mast cells in human lung possess -receptors on their surface [28]. -Adrenoreceptor agonists have been shown to inhibit the anaphylactic release of mediators, including histamine, cLTs (LTC4, LTD4, and LTE4) and TxA2 [29]. Histamine has been shown to be involved in PIB in asthmatic patients [30]. Our previous animal studies have demonstrated that TxA2, cLTs and PAF are important in the PIB [26,31,32]. These findings suggest that inflammatory mediators contribute to the development of PIB [26,31,32]. This study was designed to explore whether in vivo airway eosinophil accumulation enhances or reduces antigen-induced bronchoconstriction and/or the following PIB, both of which are mediated by lipid mediators [26,31,32].
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2. Materials and methods 2.1. A model of eosinophilic bronchitis Male, albino, Hartley strain guinea pigs weighing 200 g were obtained from Sankyou Laboratory Service (Toyama, Japan). They were transnasally treated with 75 g/kg of polymyxin-B twice per week for a total of 3 weeks. For each procedure, animals were mildly anaesthetized by diethyl ether. Control animals were administered normal saline as the vehicle in the same manner. 2.2. Passive sensitization of guinea pigs Guinea pig homocytotropic antiserum was made by the modified method of Santives et al. [33]. Briefly, 500 g of ovalbumin (OA) was emulsified in Freund’s complete adjuvant and injected intradermally into each guinea pig at multiple sites. A booster dose was prepared and administered in the same manner 2 weeks later. Serum was collected from each animal 2 weeks after the booster dose, pooled, and kept frozen until use. The antibody titer of this serum was 1:12 800, 1:6400, and 1:512, as estimated by passive cutaneous anaphylaxis at 4 h, 24 h, and 7 days, respectively. Normal guinea pigs were passively sensitized with 1 mL of antiserum/kg intraperitoneally immediately after the last administration of polymyxin-B or vehicle. 2.3. Installation of artificial respiration Guinea pigs were anesthetized by an intraperitoneal injection of 75 mg/kg sodium pentobarbital and placed in the supine position (Fig. 1) 24 h after the last administration of polymyxin-B or normal saline, followed by passive sensitization. The trachea was cannulated with a polyethylene tube (2.5 mm outside diameter and 2.1 mm inside diameter). The animals were artificially ventilated by using a small animal respirator (Model 1680, Harvard Apparatus Co., South Natick, MA, USA) adjusted to a tidal volume of 10 mL/kg at a rate of 60 strokes/min. The changes in lung resistance to inflation, the lateral pressure of the tracheal tube (pressure at the airway opening; Pao, cm of H2O), were measured by using a pressure transducer (Model TP-603T, Nihon Koden Kogyo Co., Ltd., Tokyo, Japan). Because we [34] demonstrated that the changes in the Pao after an antigen challenge in passively sensitized guinea pigs treated with and without antihistamines and after an inhalation of LTC4 represented the averages of the changes in pulmonary resistance and reciprocal dynamic lung compliance, we used change in the Pao as an overall index of the bronchial response to bronchoactive agents. When all procedures were completed, the animals were administered diphenhydramine hydrochloride (60 mg/kg intraperitoneally) to block the action of histamine. By clamping the outlet port of the respirator, we overinflated the animals by two times tidal volume for two breaths [34]. All the animal procedures in this study complied with the standard set out in the guidelines for the care and use of laboratory animals in Takara-machi campus of Kanazawa University.
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Fig. 1. Design of the experimental system. An anaesthetized guinea pig was placed in the supine position and the trachea was cannulated with polyethylene tube. The animal was artificially ventilated by a small animal respirator and nebulized with ovalbumin and propranolol. The Pao was continuously recorded to estimate the OA and the propranolol-induced bronchoconstriction by a X–Y recorder.
2.4. Experimental protocols 2.4.1. Study 1: Influence of airway eosinophil accumulation on antigen- and propranololinduced bronchoconstriction The guinea pigs were assigned into two groups (Fig. 2): control group and polymyxinB-treated group (n ⫽ 8 in each group). Fifteen minutes after the installation of artificial respiration, when the Pao had stabilized, the passively sensitized animals were challenged with nebulized OA dissolved in physiologic saline (1 mg/mL) without interrupting the constant ventilation. The OA aerosol was generated for 30 s by using an ultrasonic nebulizer developed for small animals at our institution [35]. The amount of aerosol was 15.2 L/min, and 46.4% of the aerosol was deposited in the lung as measured by the radio-aerosol technique [35]. Twenty minutes after the OA provocation, 10 mg/mL of propranolol was inhaled for 30 s. 2.4.2. Study 2: Influence of single transnasal administration of polymyxin-B on bronchial responsiveness to inhaled methacholine The guinea pigs were assigned into two groups: control group and polymyxin-B treated group (n ⫽ 8 in each group). Polymyxin-B in a dose of 75 g/kg or saline as the vehicle was transnasally administered 24 h before this experiment. Fifteen minutes after the installation of artificial respiration, when the Pao had stabilized, ascending doses of methacholine (12.5, 25, 50, and 100 g/mL) were inhaled for 20 s through the nebulizer at intervals of 5 min. 2.5. Analysis of BAL cells Cell composition of BAL fluid was conducted 15 min after the inhalation of propranolol in Study 1, and 5 min after the inhalation of 100 g/mL of methacholine in Study 2. Each
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Fig. 2. Experimental protocols. Group 1, passively sensitized guinea pigs treated with repeated intranasal administration of saline; group 2, passively sensitized guinea pigs treated with repeated intranasal administration of polymyxin-B; group 3, nonsensitized guinea pigs treated with single transnasal administration of saline; group 4, nonsensitized guinea pigs treated with single transnasal administration of polymyxin-B.
BAL fluid was compared between animals treated with polymyxin-B and saline. Ten milliliters of sterile saline were instilled, at room temperature, through a polyethylene cannula introduced into the trachea, and recovered immediately. Following this manner, the same procedure was repeated; a total of 20 mL of sterile saline was used for BAL. After filtrating the fluid through a double layer of Dacron nets, cells were centrifuged at 1000 rev./min for 12 min and resuspended in 3 mL of RPMI-1640 (Grand Island Biological Co., Grand Island, NY, USA). Following dilution with an equal volume of Tu¨rk solution, the number of cells was counted in a Bu¨ker chamber. The fluid was then resuspended with RPMI-1640 to make a cell suspension containing 2 ⫻ 105 cells/mL. Smears for differential cell counts were prepared by cytocentrifugation (Cytopin 2, Shandon Southern Products Ltd., Cheshier, UK) at 800 rev./min for 8 min. After staining with May-Gru¨nwald-Giemsa stain (Diff-Quick, American Scientific Products, McGraw Park, IL, USA), 300 cells were counted. 2.6. Chemicals The following chemicals were used: sodium pentobarbital (Abbot Laboratories, North Chicago, IL, USA), polymyxin-B sulfate (Pfizer Taito Co. Ltd., Tokyo), diethyl ether (Wako Pure Chemical IN, Osaka, Japan), OA (Sigma, St. Louis, MO, USA), diphenhydramine hydrochloride (Sigma), and dl-propranolol hydrochloride (Wako Pure Chemical Ind.)
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Fig. 3. BAL cell findings. BAL was performed 15 min after an inhalation of propranolol following an OA challenging in passively sensitized guinea pigs treated with repeated intranasal administration of saline (open bars, n ⫽ 8) or polymyxin-B (shaded bars, n ⫽ 8). Error bars indicate mean ⫾ SEM. *p ⬍ 0.05 and **p ⬍ 0.01 compared with the saline group.
2.7. Statistical analysis One-way ANOVA was used for analyzing difference in baseline Pao value or each cell component in BAL fluid between groups. The degree of bronchoconstriction was evaluated using a time course curve for the percentage increase in the Pao from the preantigen challenge value induced by antigen provocation or propranolol inhalation, and was analyzed by repeated measure ANOVA. These analyses were performed using the software StatView 4.02 (Abacus Concepts, Berkeley, CA, USA). A p value of less than 0.05 was considered statistically significant, and values were expressed as the mean ⫾ SEM.
3. Results 3.1. Study 1: Influence of airway eosinophil accumulation on antigen- and propranololinduced bronchoconstriction The total number of effector cells recovered per milliliter in BAL fluid was significantly different ( p ⬍ 0.05) between treatment with polymyxin-B (8.8 ⫾ 2.9 ⫻ 105 cells/mL) and vehicle (saline) (5.3 ⫾ 0.7 ⫻ 105 cells/mL). Bronchoalveolar cell findings are shown in Fig. 3. The proportion of eosinophils in BAL fluid was significantly increased in guinea pigs treated with polymyxin-B compared with the saline. There were no significant differences between the groups about the proportion of neutrophils and lymphocytes in BAL fluid. Baseline Pao value before OA challenge did not differ significantly between animals treated with polymyxin-B (10.2 ⫾ 0.6 cm of H2O) and saline (10 ⫾ 1.3 cm of H2O). Fig. 4 shows time course of the percentage increases in the Pao after OA provocation followed by propranolol inhalation. The time-course curve for the percentage increases in the Pao after the OA challenge or the following propranolol inhalation did not differ between the two groups.
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Fig. 4. Time course of percentage increases in Pao after an aerosolized OA challenge followed by an inhalation of propranolol in passively sensitized guinea pigs treated with repeated intranasal administration of saline (open circle, n ⫽ 8) or 75 g/kg polymyxin-B (closed circle, n ⫽ 8). Vertical bars represent SEM. NS ⫽ not significant.
3.2. Study 2: Influence of single transnasal administration of polymyxin-B on bronchial responsiveness to inhaled methacholine The total number of effector cells recovered per milliliter in BAL fluid was not significantly different between treatment with polymyxin-B (13.8 ⫾ 1.5 ⫻ 105 cells/mL) and vehicle (saline) (10.1 ⫾ 1.9 ⫻ 105 cells/mL). Bronchoalveolar cell findings are shown in Fig. 5. There was no significant difference in the proportion of neutrophils, eosinophils or lymphocytes in BAL fluid between the groups. Fig. 6 shows the percentage of increases in the Pao caused by inhalation of ascending doses of methacholine. Baseline Pao value before methacholine inhalation did not differ significantly between animals treated with polymyxicin B (9.4 ⫾ 0.3 cm of H2O) and saline (9.2 ⫾ 0.2 cm of H2O). Percentage increases in the Pao produced by each dose of methacholine did not differ between the two groups.
Fig. 5. BAL cell findings. BAL was performed 5 min after final inhalation of methacholine in nonsensitized guinea pigs treated with repeated intranasal administration of saline (open bars, n ⫽ 8) or polymyxin-B (shaded bars, n ⫽ 8).
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Fig. 6. Percentage of increases in Pao after inhalation of each dose of methacholine in nonsensitized guinea pigs treated with repeated intranasal administration of vehicle (open circle, n ⫽ 8) or 75 g/kg polymyxin-B (closed circle, n ⫽ 8). Vertical bars represent SEM. NS ⫽ not significant.
4. Discussion In this study, airway eosinophil accumulation induced by repeated transnasal administration of polymyxin-B did not alter antigen-induced bronchoconstriction or subsequent PIB in passively sensitized guinea pigs pretreated with diphenhydramine in vivo. Recently, chronic desquamative eosinophilic bronchitis has been considered to be a fundamental feature of bronchial asthma [36,37] and significant correlation between the degree of BHR and airway eosinophil accumulation in stable asthmatics has been demonstrated [3–5]. The role of eosinophil-derived mediators in the development of BHR has also been postulated because of the significant correlation between the concentration of major basic protein and the number of eosinophils in BAL fluid in asthmatics [3]. However, a recent study indicates that eosinophil-derived major basic protein inhibits antigen-induced BHR in sensitized guinea pigs [38]. Sanjar and colleagues [11,12] also have reported that there is no significant increase in the airway reactivity after PAF challenge or antigen challenge by aerosol in guinea pigs, despite the airway eosinophil accumulation. Sasaki et al. [39] have shown that interleukin-5, which is known as a chemotactic factor for eosinophils, does not increase bronchial responsiveness in guinea pigs, and others have failed to demonstrate the relationship between the BHR and the number of eosinophils in biopsied bronchial mucosa in asthmatics [9,10,13]. Moreover, we have shown that bronchial responsiveness to methacholine is not increased in patients with bronchodilator-resistant cough with sputum eosinophilia and cough hypersensitivity (atopic cough) [21] and eosinophilic pneumonia [22], despite the increase of eosinophils in biopsied bronchial mucosa. Gibson et al. [23] have also shown that sputum eosinophilia is not associated with BHR. Although previous investigators demonstrated the generation of cLTs and PAF by eosinophils in vitro [40 – 42], others indicated that some eosinophil-derived factors may alter the allergic reaction by metabolizing or inactivating chemical mediators: histaminase and arylsulfatase may limit or possibly terminate the allergic reaction [14 –20]. These findings described above suggest that eosinophilic inflammation may not be
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essential for the development of bronchial asthma and BHR. The exact role of eosinophils in bronchoconstriction mediated by intrinsic mediators in vivo also remains unclear. Thus, we conducted this study to explain the influence of airway eosinophil accumulation on antigen-induced bronchoconstriction and PIB in passively sensitized guinea pigs in vivo. Polymyxin-B used in this study is a mast cell degenerating agent [43], and is known to promote the accumulation and activation of eosinophils [44]. We recently developed an animal model of eosinophilic bronchitis induced by polymyxin-B and have demonstrated the increase in the number of accumulated eosinophils in the mucosa and the injury to airway epithelium in trachea, large and medium bronchi, but not in the alveolar region or the lung structure [45]. The degree of activation of eosinophils recruited in this study has remained unknown, because we did not measure it. However, we consider that eosinophils recruited by polymyxin-B are activated in vivo as Davis et al. [46] have shown it. It is well known that most of the known mediators are involved in allergic bronchoconstriction [47], and our previous animal studies have also demonstrated the involvement of lipid mediators in pathogenesis of PIB [26,31,32]. Although this animal model of PIB can not evaluate dose response relation to propranolol, it can assess the effect of propranolol in a time-course study. As the mediator mechanism appears to play an important role in both antigen-induced bronchoconstriction and PIB, our animal model for PIB after antigeninduced bronchoconstriction is considered able to evaluate the influence of various factors on bronchoconstriction mediated by chemical mediators, except for histamine. In the present study, a 3-week transnasal administration of polymyxin-B did not alter antigen-induced bronchoconstriction or after PIB, despite the increased number of airway eosinophils as proven in BAL fluid. We also showed that a single transnasal administration of polymyxin-B did not affect bronchial responsiveness to inhaled methacholine. As our previous study regarding airway eosinophil accumulation revealed that repeated transnasal administration of polymyxin-B does not affect bronchial responsiveness to inhaled histamine despite increased infiltration of eosinophils in intrapulmonary small airways [45], we can evaluate the influence of airway eosinophil accumulation on bronchoconstrictor response mediated by intrinsic lipid mediators in this animal model. Although additional remarks may be done for the sensitization and challenge with OA and the association of polymyxin B plus antigen challenge, another previous study indicated that antigen challenge does not cause allergic airway reaction in nonsensitized animals [26]. As mentioned previously, our results imply that eosinophils accumulated by repeated transnasal administration of polymyxin-B in vivo do not potentiate or prevent chemical mediator release such as TxA2, cLTs, and PAF, which has been proven to be involved in both the antigen-induced bronchoconstriction and the following PIB in our animal model [26,31, 32]. We conclude from this study that in vivo eosinophil accumulation in the airways does not influence releasability of mediators from effector cells or inactivation of released mediators. Further studies may be required to explore the influence of eosinophil accumulation for histamine releasability in allergic bronchoconstriction and the role of mast cells, neutrophils, and other cells in bronchoconstriction mediated by various intrinsic mediators in vivo.
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