Platelet-Activating Factor Receptor Develops Airway Hyperresponsiveness Independently of Airway Inflammation in a Murine Asthma Model This information is current as of September 24, 2021. Satoshi Ishii, Takahide Nagase, Hideo Shindou, Hajime Takizawa, Yasuyoshi Ouchi and Takao Shimizu J Immunol 2004; 172:7095-7102; ; doi: 10.4049/jimmunol.172.11.7095 http://www.jimmunol.org/content/172/11/7095 Downloaded from References This article cites 69 articles, 31 of which you can access for free at: http://www.jimmunol.org/content/172/11/7095.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! 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The Journal of Immunology Platelet-Activating Factor Receptor Develops Airway Hyperresponsiveness Independently of Airway Inflammation in a Murine Asthma Model1 Satoshi Ishii,2*§ Takahide Nagase,† Hideo Shindou,*§ Hajime Takizawa,† Yasuyoshi Ouchi,‡ and Takao Shimizu*§ Lipid mediators play an important role in modulating inflammatory responses. Platelet-activating factor (PAF) is a potent proin- flammatory phospholipid with eosinophil chemotactic activity in vitro and in vivo. We show in this study that mice deficient in PAF receptor exhibited significantly reduced airway hyperresponsiveness to muscarinic cholinergic stimulation in an asthma model. However, PAF receptor-deficient mice developed an eosinophilic inflammatory response at a comparable level to that of wild-type mice. These results indicate an important role for PAF receptor, downstream of the eosinophilic inflammatory cascade, in reg- Downloaded from ulating airway responsiveness after sensitization and aeroallergen challenge. The Journal of Immunology, 2004, 172: 7095–7102. ronchial asthma is a complex disease of the lung char- models of asthma, because IL-5-deficient BALB/c mice partially acterized by reversible airway obstruction, chronic air- developed AHR by OVA sensitization/challenge in the absence of way inflammation, and airway hyperresponsiveness airway inflammation (9). Conversely, IL-10-deficient C57BL/6 B3 (AHR) to nonspecific stimuli. The progression of airway inflam- mice failed to develop AHR even in the presence of robust airway http://www.jimmunol.org/ mation involves several cell types, including CD4ϩ Th2 cells, eo- inflammation (10). sinophils, and mast cells (1). The immunopathogenic role of Th2 Platelet-activating factor (PAF; 1-O-alkyl-2-acetyl-sn-glycero- cells is suggested by the roles of their products, such as IL-4, IL-5, 3-phosphocholine) is a potent phospholipid mediator with various and IL-13 in the recruitment and activation of the primary effector biological activities besides platelet activation (11). PAF acts by cells of the allergic response, eosinophils and mast cells. Activa- binding to a G protein-coupled seven-transmembrane receptor tion of these cells results in the release of many inflammatory (12–16). PAF has long been implicated in the pathophysiological mediators that seem to induce AHR individually or coordinately mechanisms of asthma (17), because exogenous PAF closely mim- (2, 3), although the precise molecular mechanisms predisposing to ics many of the clinical features of asthma, including AHR (18, the development of AHR in asthmatics are largely unknown. The 19), bronchoconstriction (18), tracheal fluid secretion (20), and by guest on September 24, 2021 hypothesis that airway inflammation is responsible for AHR is airway microvascular leakage (21) in animals and humans. PAF is based on the finding of a significant relationship between the pa- detected in bronchoalveolar lavage (BAL) fluid from asthmatic rameters of airway inflammation and AHR (4, 5) and on the ob- patients but not from nonallergic subjects (22). Eosinophils and servation that inhaled steroids reduce both airway inflammation mast cells activated in asthmatic airways may be the cellular ori- and AHR (6, 7). However, a number of studies in asthmatic pa- gins of PAF, because these cells are known to produce PAF in tients have cast doubt on the requirement of airway inflammation response to various stimuli in vitro (23, 24). Furthermore, PAF was for AHR (see review in Ref. 8). In addition, dissociation of AHR reported to be a potent chemotactic factor for eosinophils (25) and from airway inflammation has also been reported in some mouse to induce eosinophil degranulation in vitro (26). By using PAF receptor-deficient ( pafrϪ/Ϫ) and PAF receptor-overexpressing mice, we have previously demonstrated that PAF plays a critical Departments of *Biochemistry and Molecular Biology, †Respiratory Medicine, and ‡Geriatric Medicine, Faculty of Medicine, University of Tokyo, Tokyo, Japan; and role in anaphylaxis and acute injury in the lung (27, 28), suggest- §Core Research for Evolutional, Science and Technology, Japan Science and Tech- ing that PAF mediates early-phase responses of allergy and in- nology Agency, Saitama, Japan flammation in the tissue. However, the importance of PAF in the Received for publication August 26, 2003. Accepted for publication March 26, 2004. development of the allergen-induced AHR and chronic inflammation The costs of publication of this article were defrayed in part by the payment of page associated with asthma has not yet been investigated in pafrϪ/Ϫ mice. charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. To define the role of PAF in the late-phase responses of allergy, we 1 This work was supported in part by Grants-in-Aid from the Ministry of Education, used an established murine asthma model, where mice were immu- Science, Culture, Sports and Technology of Japan (to T.S., T.N., and S.I.), Grants- nized with aluminum hydroxide adjuvant-adsorbed OVA and chal- in-Aid for Comprehensive Research on Aging and Health (to T.N. and S.I.) and for lenged with aerosolized OVA. In this study, we describe that pafrϪ/Ϫ Research on Allergic Disease and Immunology (to S.I.) from the Ministry of Health, Labour and Welfare of Japan, and also grants from the Yamanouchi Foundation for mice, sensitized and challenged with OVA, displayed reduced AHR Research on Metabolic Disorders (to T.N. and S.I.), the Kanae Foundation for Life despite a significant eosinophilic airway inflammatory response. PAF and Socio-medical Science, and the Uehara Memorial Foundation (to S.I.). may contribute to AHR in asthmatics independently of the eosino- 2 Address correspondence and reprint requests to Dr. Satoshi Ishii, Department of philic airway inflammation. Biochemistry and Molecular Biology, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo, Tokyo 113-0033, Japan. E-mail address: [email protected] tokyo.ac.jp Materials and Methods 3 Abbreviations used in this paper: AHR, airway hyperresponsiveness; PAF, platelet- Mice activating factor; BAL, bronchoalveolar lavage; LT, leukotriene; PAS, periodic acid- Ϫ/Ϫ ϫ Schiff; RL, total lung resistance; EC200RL, effective concentration of methacholine pafr mice were produced on a mixed C57BL/6 129/Ola genetic back- Ϫ/Ϫ required to double the basal RL. ground as described previously (27). In the present study, pafr mice and Copyright © 2004 by The American Association of Immunologists, Inc. 0022-1767/04/$02.00 7096 MODULATION OF AIRWAY RESPONSIVENESS BY PAF RECEPTOR the corresponding wild-type ( pafrϩ/ϩ) control mice have been backcrossed inhibitor mixture (Complete; Roche, Mannheim, Germany) at room tem- for 6–10 generations onto a BALB/c genetic background. The animals perature. The initial lavage was instilled and retrieved one time, whereas were maintained in a light-dark cycle with light from 7:00 a.m. to 8:00 p.m. the second lavage was instilled twice. This procedure allowed for a greater at 22°C. Mice were fed with a standard laboratory diet and water ad libi- number of lung washes with less diluent. In total, ϳ1.6 ml of BAL fluid tum. All of the mice in this study were used under a protocol approved by was consistently recovered. The sample was centrifuged at 1000 rpm for 10 the University of Tokyo Ethics Committee for Animal Experiments. min at 4°C, and the supernatant was collected and stored at Ϫ80°C. The During the course of the backcrossing, we selected mice homozygous cell pellet was resuspended in 200–250 ␮l of cold saline containing 0.1% for the intact allele of the group IIA phospholipase A2 gene that is linked fatty acid-free BSA (Serologicals Proteins, Kankakee, IL). After an appro- to the PAF receptor gene on murine chromosome 4 (29, 30). The genetic priate dilution (2- to 20-fold) of the cell suspension with Turk solution distance between these genes is ϳ4.3 cM. Both C57BL/6 and 129/Ola (Mutoh Chemical, Tokyo, Japan), the total cell number was counted with ϫ inbred mice are deficient in group IIA phospholipase A2 due to a congenital a hemocytometer. Slides of BAL fluid cells were prepared by placing 3 disruption of the gene, whereas BALB/c inbred mice have an intact gene 105 cells into a cytocentrifuge (Cytospin 3; Shandon, Pittsburgh, PA) at for group IIA phospholipase A2 (29, 31). Thus, our selection was able to 350 rpm for 2 min, and staining with Diff-Quik (International Reagents, exclude the possible effects of group IIA phospholipase A2 deficiency, Kobe, Japan).