Airway Responsiveness in Transgenic Mice ... - ATS Journals

Airway Responsiveness in Transgenic Mice Overexpressing Platelet-activating Factor Receptor Roles of Thromboxanes and Leukotrienes TAKAHIDE NAGASE, SATOSHI ISHII, HIROFUMI KATAYAMA, YOSHINOSUKE FUKUCHI, YASUYOSHI OUCHI, and TAKAO SHIMIZU Department of Geriatrics, Department of Biochemistry, Faculty of Medicine, University of Tokyo, Tokyo, Japan

Platelet-activating factor (PAF) is a potent proinflammatory compound potentially involved in the pathogenesis of inflammatory disorders, including bronchial asthma. To elucidate the pathophysiologic roles of PAF in bronchial asthma, we studied airway responsiveness in transgenic mice overexpressing PAF receptor. In the transgenic mice, PAF-induced airway smooth muscle contraction was demonstrated by physiologic and morphometric analyses, whereas there was no significant response in the littermate control group. The PAF-elicited bronchoconstriction in the transgenic mice was significantly reduced not only by a PAF receptor antagonist (WEB-2086) but also by a thromboxane synthesis inhibitor (indomethacin or ozagrel), an inhibitor of 5-lipoxygenase-activating protein (MK886), or a cysteinyl leukotriene (LT) antagonist (pranlukast). LTB4 receptor antagonist (ONO-4057), however, had no effect on the PAF-induced responses. The transgenic mice showed a bronchial hyperreactivity to methacholine challenge, which was also inhibited by pretreatment with either thromboxane synthesis inhibitor or cysteinyl LT antagonist. These observations suggest that both thromboxane A2 and cysteinyl LTs (LTC4, LTD4, and LTE4) are involved in the bronchial responses to PAF or cholinergic stimulus in mice. The transgenic mice overexpressing PAF receptor may provide an appropriate model to study various PAF-related lung diseases, including bronchial asthma. Nagase T, Ishii S, Katayama H, Fukuchi Y, Ouchi Y, Shimizu T. Airway responsiveness in transgenic mice overexpressing platelet-activating factor receptor: roles of thromboxanes and leukotrienes. AM J RESPIR CRIT CARE MED 1997;156:1621–1627.

Platelet-activating factor (PAF) is a phospholipid mediator that has potent inflammatory properties (1–3). PAF mediates its biologic effects via activation of a G-protein-coupled seven transmembrane receptor (2–4). PAF receptor cDNA and genes have been cloned from various species, including guinea pigs and humans (4–10). Recently, we have established transgenic mice ubiquitously overexpressing PAF receptor by selecting the b-actin promotor and cytomegalovirus enhancer (11). Northern blot analysis showed the expression of the transgene in the heart, skeletal muscle, eye, skin, trachea, and aorta (11). It has recently been postulated that PAF may have a potential pathophysiologic role in bronchial asthma (12, 13). In the lung of human asthmatics, increases in PAF receptor mRNA expression level have been demonstrated (14). It has also been reported that deficiency of plasma PAF acetylhydrolase is associated with respiratory impairment in asthmatic children (15, 16). The exogenous PAF administration enhances bronchial hyperresponsiveness, which is a major feature of asthma

(Received in original form March 5, 1997 and in revised form July 9, 1997) Supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture, Japan. Correspondence and requests for reprints should be addressed to Dr. T. Nagase, Department of Geriatrics, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, Japan, 113. Am J Respir Crit Care Med Vol 156. pp 1621–1627, 1997

(17). However, the exact pathophysiologic roles of PAF in the pathogenesis of bronchial asthma remain to be clarified. In the current study, we hypothesized that eicosanoids, including thromboxanes and leukotrienes (LTs), might be involved in PAF-related airway hyperresponsiveness. To test this hypothesis, we investigated the effects of eicosanoid blockade on airway responsiveness in transgenic mice, which genetically overexpress PAF receptor.

METHODS Transgenic Mice Transgenic mice were established in which the guinea pig PAF receptor cDNA was placed under the regulation of the chicken b-actin promotor as previously described (11). Mice were housed in an air-conditioned room at 258 C and fed a standard laboratory diet and water ad libitum. Male transgenic mice were mated to BDF1 female mice. Offspring were genotyped at 4 wk of age. For genotyping, genomic DNAs were isolated from biopsied tail and subjected to 30 cycles of PCR amplification (1 min at 948 C; 2 min at 558 C; 2 min at 728 C). The primers specific to the guinea pig PAF receptor cDNA were: forward 59-ACCACACTCCTGTCAATC-39 and reverse 59-TCAGGATCAGGTCATGAT-39. The PCR product consisted of 290 bp, which was confirmed by Southern blot analysis. The transgenic mice and their littermate controls (13 to 24 wk old) were used in the current study.

Animal Preparation Animals were anesthetized intraperitoneally with pentobarbital sodium (25 mg/kg) and ketamine hydrochloride (25 mg/kg) in combina-

1622

AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE

tion and then paralyzed intraperitoneally with pancuronium bromide (0.3 mg/kg). Anesthesia and paralysis were maintained by supplemental administration of 10% of the initial dose every hour. After tracheostomy, an endotracheal metal tube (inside diameter, 1 mm; length, 8 mm) was inserted into the trachea. Animals were mechanically ventilated (Model 683; Harvard Apparatus, South Natick, MA) with tidal volumes of 8 ml/kg and frequencies of 2.5 Hz. The thorax was widely opened by means of midline sternotomy, and a positive end-expiratory pressure of 3 cm H2O was applied by placing the expired line underwater. During the experiments, oxygen gas was continuously supplied to the ventilatory system. Under these ventilatory conditions, arterial pH, PO2, and PCO2 were 7.35 to 7.45, 100 to 180, and 30 to 45 mm Hg, respectively, at the end of experiments. A heating pad was used to maintain the body temperature of animals. Tracheal pressure was measured with a piezoresistive microtransducer (8510B-2; Endevco, San Juan Capistrano, CA) placed in the lateral port of the tracheal cannula. Tracheal flow was measured by means of a Fleisch pneumotachograph (Model no. 00000; Metabo SA, Lausanne, Switzerland). All signals were amplified, filtered at a cutoff frequency of 100 Hz, and converted from analog to digital with a converter (DT2801-A; Data Translation Inc., Marlborough, MA). The signals were sampled at a rate of 200 Hz and stored on an IBM-AT compatible computer. Lung resistance (RL) was measured as previously described (11, 18–20).

VOL 156

1997

Figure 1. Time course of responses to 10 mg/kg PAF administration in transgenic mice (n 5 9) and in littermate control mice (n 5 9). Asterisks indicate p , 0.01 compared with control mice. R L 5 lung resistance.

Airway Responsiveness to PAF Administration PAF was dissolved in the vehicle (0.25% bovine serum albumin in saline) at a concentration of 3.33 mg/ml and administered at a dose of 10 mg/kg. This dose was chosen because the administration of . 10 mg/kg PAF caused severe arrhythmia or cardiac failure in the transgenic mice in a preliminary experiment. After baseline measurements, the vehicle of PAF and subsequent 10 mg/kg PAF were administered via the jugular vein. Each solution was injected at a volume of 3 ml/kg. After the bolus of 10 mg/kg PAF, measurements were made for as long as 10 min.

Morphometric Study In four animals from the transgenic and the control groups, we studied the PAF-induced responses using morphometric techniques. Ten minutes after PAF administration, the lungs were removed intact and frozen with liquid nitrogen. A constant transpulmonary pressure of 3 cm H2O was maintained during freezing by delivering constant flow into the trachea. Frozen lungs were fixed in Carnoy’s solution (60% ethyl alcohol, 30% chloroform, 10% acetic acid) at 2708 C for 18 h. Progressively increasing concentrations of ethanol at 2208 C were then substituted for the Carnoy’s solution until 100% ethanol was reached. The tissue was maintained at 2208 C for 4 h, warmed to 48 C for 12 h, and then allowed to reach and remain at room temperature for 2 h. After fixation, the tissue blocks obtained from midsagittal slices of the lungs were embedded in paraffin. Blocks were cut 4 mm thick using a microtome. Tissues were stained with hematoxylin-eosin. We assessed tissue shrinkage, and subsequent measurements were corrected for shrinkage. Airway constriction was assessed by measuring the length of the epithelial basement membrane (Pbm) and the area (Abm) it circumscribed by projecting microscopic images onto a digitizer by means of a drawing attachment fixed to the microscope. The ideal area of the lumen of the relaxed airway (Abm*) was then calculated as 2

Abm* = Pbm ⁄ 4π

(1)

and the degree of constriction (Abm/Abm*) was derived (21). The area circumscribed by the outer border of adventitia (Ao) was also measured and the area of the airway wall (WA) was calculated by the difference between Ao and Abm. We normalized WA to the relaxed area to adjust for differences in the airway size. To assess whether

airways were cut in cross section, the maximal diameter of the airway (D1) and the diameter at the widest point perpendicular to this axis (D2) were determined. We analyzed airways only with a ratio of D2/D1 . 0.5.

Effects of Drugs on PAF-induced Responses Two minutes prior to the bolus of 3 ml/kg solution containing 10 mg/kg PAF, the transgenic mice were pretreated with one of the following solutions: (1) vehicle as controls (n 5 9), (2) 1 mg/kg WEB-2086 (PAF receptor antagonist, n 5 4), (3) 20 mg/kg indomethacin (n 5 5), (4) 200 mg/kg ozagrel (thromboxane synthase inhibitor, n 5 5), (5) 20 mg/kg pranlukast (cysteinyl LT receptor antagonist, n 5 4), (6) 20 mg/kg MK-886 (5-lipoxygenase-activating protein inhibitor, n 5 5), and (7) 25 mg/kg ONO-4057 (LTB4 receptor antagonist, n 5 4). Littermate control mice were pretreated with vehicle (n 5 9). After the pretreatment with each solution, we made a measurement at baseline. There were no significant differences in baseline RL between each group. After the PAF administration, measurements were made for as long as 10 min.

Effects of Drugs on Airway Responsiveness to Methacholine Administration Two minutes prior to the nebulization, transgenic mice were pretreated with one of the following solutions: (1) vehicle as controls (n 5 8), (2) 200 mg/kg ozagrel (thromboxane synthase inhibitor, n 5 6), and (3) 20 mg/kg pranlukast (cysteinyl LT receptor antagonist, n 5 6). Littermate control mice were pretreated with vehicle (n 5 8). At the start of the protocol, two deep inhalations (three times tidal volume) were delivered to standardize volume history. All animals were then challenged with saline aerosol for 1 min. Aerosols were generated by an ultrasonic nebulizer (Ultra-Neb 100; DeVilbiss, Somerset, PA) and delivered through the inspiratory line into the trachea. Measurements of 10-s duration were sampled during tidal ventilation 1 min after administration of saline aerosol. This represented the baseline measurement. Then, each dose of methacholine (MCh) aerosol was administered for 1 min in a dose-response manner (0.31 to 80 mg/ml). Airway responsiveness was assessed using the concentration of MCh required to increase lung resistance to 200% of baseline values (EC200).

Figure 2. Lung histology in (A) transgenic mice and in (B) littermate control mice after 10 mg/kg PAF administration. The lungs were frozen with liquid nitrogen, fixed with Carnoy’s solution, and stained with hematoxylin-eosin. Note that PAF administration induces substantial bronchoconstriction in the transgenic mice, whereas airways seem normal in control mice. Original magnification: 340.

Nagase, Ishii, Katayama, et al.: Airway Responsiveness in Transgenic Mice Overexpressing PAF Receptor

1623

1624

AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE

VOL 156

1997

Data Analysis Comparisons of physiologic data among the experimental groups were carried out with ANOVA (Newman-Keuls test). Morphometric data were analyzed with Student’s t test. Data are expressed as mean 6 SE; p values less than 0.05 were taken as significant.

RESULTS Airway Responsiveness to PAF Administration

Figure 3. Airway constriction assessed by morphometric analysis in transgenic (n 5 4) and in control (n 5 4) mice. Abm/Abm* 5 airway narrowing normalized with the ideally relaxed airway size. Asterisk indicates p , 0.001 compared with control mice. Materials and Chemicals Materials and chemicals were obtained from the following sources. PAF was from Cayman Chemical (Ann Arbor, MI); bovine serum albumin, MCh, and indomethacin were from Sigma Chemical Co. (St. Louis, MO); pranlukast, ozagrel, and ONO-4057 were from Ono Pharmaceutical Co. (Osaka, Japan). WEB-2086 and MK-886 were gifts from Boehringer Ingelheim (Ingelheim, Germany) and Merck Frosst Canada Inc. (Pointe-Claire-Dorval, Canada), respectively.

The time course of bronchopulmonary responses to PAF administration in the transgenic and littermate control mice is shown in Figure 1. In the transgenic mice, maximal responses were observed at 7.7 6 0.8 min after intravenous administration of PAF. In contrast, no significant responses were detected in the control mice. Lung histology of the transgenic and control mice after PAF administration is shown in Figure 2. In the transgenic mice, a substantial degree of bronchoconstriction was observed, whereas airways were not constricted in the control mice. In the morphometric study, there were no significant differences in airway size between the transgenic and control mice (Pbm, 0.827 6 0.042 versus 0.824 6 0.040 mm, respectively), indicating that there were no significant biases between the groups in terms of airway selection. As shown in Figure 3, morphometric analysis showed significant bronchoconstriction in the transgenic mice. Meanwhile, there was no significant difference in airway wall thickness between the transgenic and the control mice (WA/Abm*, 0.290 6 0.013 versus 0.298 6 0.015, respectively).

Figure 4. Effects of antagonists on PAF-induced bronchopulmonary responses. Responses to PAF are expressed as %change in lung resistance. *p , 0.001 versus transgenic and ONO-4057 groups. #p , 0.01 versus control, transgenic, WEB-2086, and ONO-4057 groups.

Nagase, Ishii, Katayama, et al.: Airway Responsiveness in Transgenic Mice Overexpressing PAF Receptor Effects of Drugs on PAF-induced Responses

The effects of drugs on bronchopulmonary responses to PAF administration in the transgenic mice are summarized in Figure 4. A PAF receptor antagonist, WEB-2086, completely ablated PAF-induced responses. The PAF-elicited bronchoconstriction in the transgenic mice was significantly attenuated by a thromboxane synthesis inhibitor (indomethacin or ozagrel), a 5-lipoxygenase-activating protein inhibitor (MK-886), or a cysteinyl LT antagonist (pranlukast). In contrast, LTB4 receptor antagonist (ONO-4057) did not affect the PAF-induced responses. Effects of Drugs on Airway Responsiveness to MCh Administration

The results of MCh-challenge study are summarized in Figure 5. The transgenic mice showed a marked bronchial hyperreactivity to MCh. Either thromboxane synthesis inhibitor (ozagrel) or cysteinyl LT antagonist (pranlukast) partially, but significantly, reduced airway hyperresponsiveness to MCh in the transgenic mice.

DISCUSSION The results of the current experiments show that airway hyperresponsiveness to PAF or MCh in the transgenic mice was significantly attenuated by the blockade of thromboxane synthesis or by the antagonism of cysteinyl LT receptors. These findings suggest that thromboxanes and cysteinyl LTs (LTC4, LTD4, and LTE4) may play significant roles in PAF-related bronchopulmonary responses in mice. In the transgenic mice, marked bronchoconstriction was elicited after PAF administration. Slow and sustained increases in lung resistance were observed in the time-course study (Figure 1). On the other hand, in control mice, PAF ad-

1625

ministration did not affect lung resistance, a finding consistent with previous studies (22, 23). The present results demonstrate that murine airways overexpressing PAF receptor respond to exogenous PAF administration, resulting in a remarkable bronchoconstriction. In the morphologic study, significant airway narrowing was observed in the transgenic mice treated with PAF, whereas there was no eosinophil infiltration to airways (Figure 2). There were significant differences in morphometrically assessed bronchoconstriction between the transgenic and control mice, whereas no differences were found in terms of airway wall thickness (Figure 3). The underlying dynamic mechanism of PAF-induced airway narrowing remains to be elucidated. In guinea pigs, it has been reported that airway edema resulting from airway microvascular leakage is an important component of airway narrowing induced by PAF administration (24). In contrast, the current results suggest that PAF administration induces mainly airway smooth muscle constriction, but not airway wall thickening, in the transgenic mice. The previous study has shown that a high level of transgenic mRNA exists in the trachea of transgenic mice (11). Airway smooth muscle cells overexpressing PAF receptor may have a key role in the PAF-induced bronchopulmonary responses in the current model. The PAF-elicited bronchoconstriction in the transgenic mice was significantly attenuated not only by PAF receptor antagonist (WEB-2086) but also by a thromboxane synthesis inhibitor (indomethacin or ozagrel), a 5-lipoxygenase-activating protein inhibitor (MK-886), or a cysteinyl LT antagonist (pranlukast). LTB4 receptor antagonist (ONO-4057), however, had no effects on the PAF-induced bronchoconstriction (Figure 4). One of the possible mechanisms to explain this observation is that PAF-elicited responses may be enhanced by eicosanoids, including thromboxane A2 and cysteinyl LTs (LTC4, LTD4, and LTE4). The coupling of PAF and PAF re-

Figure 5. Effects of antagonists on methacholine-induced bronchopulmonary responses. Responsiveness is expressed as the concentration of methacholine required to double lung resistance (EC 200). *p , 0.01 versus transgenic group; #p , 0.01 versus transgenic, p , 0.05 versus ozagrel and pranlukast groups.

1626

AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE

ceptor might be involved in the releases of thromboxane A2 and cysteinyl LTs, which are also potent mediators. Recently, it has been postulated that PAF may act as a second messenger in the release of eicosanoids (1, 25). From the study using 5-lipoxygenase deficient mice, PAF has been shown to evoke the lethal shock in vivo by releasing LTs (26). Because of the substantial degree of inhibitory effects of antagonists, the direct roles of PAF in bronchoconstriction may be proportionally smaller than those of thromboxane A2 and cysteinyl LTs. The complex effects of PAF, thromboxanes, and LTs on the bronchopulmonary tree may result in the slow and sustained bronchoconstriction as shown in Figure 1. In marked contrast, it seems that PAF-induced airway narrowing is not mediated by LTB4, which is a chemotactic mediator but has little role in bronchoconstriction (27). Blockade of either thromboxane or cysteinyl LTs caused more than 80% inhibition of the response to PAF in the transgenic mice. This finding suggests that blocking thromboxane synthesis reduces the potent actions of LTs and vice versa. Possible explanation is that thromboxane A2 per se may mediate biologic effects of LTs and vice versa. It has been reported that PAF may be involved in bronchial hyperresponsiveness in various species, including humans (12, 13, 18). Recently, it was demonstrated that a specific PAF receptor antagonist, Y-24180, reduces bronchial hyperresponsiveness to MCh in patients with asthma (29). In the current transgenic mice, increases in airway responsiveness to MCh were also observed compared with the littermate control mice (Figure 5). Pretreatment with either thromboxane synthesis inhibitor (ozagrel) or cysteinyl LT antagonist (pranlukast) significantly decreased bronchial responsiveness in the transgenic mice, suggesting that both thromboxane A2 and cysteinyl LTs are involved in hyperresponsiveness related to the PAF receptor overexpression. A potential mechanism is that MChstimulated release of PAF may result in overproduction of thromboxane A2 and cysteinyl LTs, which subsequently display the contractile activity on the airway smooth muscle (30). Overexpression of PAF receptor may have an important role in the pathogenesis of bronchial hyperreactivity, one of the major characteristics of asthma. It is suggested that hereditary factors contribute to the etiology of asthma. The inheritance pattern indicates that a number of complex genes are involved in the pathogenesis of bronchial asthma (31). Recently, murine models of asthma have been used to identify individual genes involved in airway hyperresponsiveness (32). On the basis of the reports that PAF can mimic features of bronchial asthma (12, 13, 17, 24), genes that regulate the function and metabolism of PAF may be potentially associated with the pathogenesis of asthma. The PAF-related genes include the PAF receptor gene (10, 33) and genes encoding PAF-metabolic enzymes such as PAF acetylhydrolases (34, 35). In human asthmatics, PAF receptor mRNA expression is increased in the lung, whereas deficiency of serum PAF acetylhydrolase is correlated with the severity of asthma in children (14–16). The current transgenic mice, which genetically overexpress PAF receptor throughout development and life, may be useful to study the genetic contribution of PAF to the etiology of asthma. It is postulated that PAF has pleiotropic and pathophysiologic effects on various tissues and organs other than the respiratory system (1–3). PAF exerts its actions at concentrations as low as 10212 M in some systems and almost always by 1029 M as an intercellular messenger (1). It is also suggested that PAF may be an intracellular messenger as well as an intercellular messenger (1, 25). Recently, it has been demonstrated that the transgenic mice overexpressing PAF receptor exhibit an in-

VOL 156

1997

creased lethality to lipopolysaccharide, bacterial endotoxin (11). Cutaneous disorders, including melanogenesis and melanocytic proliferation, were observed and melanoma arose in some aged transgenic mice (11). The current transgenic mice may provide novel insights to study the pathophysiologic roles of PAF and PAF receptor in vivo. In summary, the transgenic mice overexpressing PAF receptor demonstrated airway smooth muscle contraction in response to exogenous PAF administration. The PAF-elicited bronchoconstriction in the transgenic mice was significantly reduced by the blockade of thromboxane or LT synthesis or by the antagonism of cysteinyl LT (but not LTB4) receptors. Airway hyperresponsiveness to MCh in the transgenic mice was significantly attenuated by either thromboxane synthesis inhibitor or cysteinyl LT antagonist. These observations suggest that both thromboxanes and LTs are involved in the mechanism of PAF-induced bronchial responses in mice. The transgenic mice overexpressing PAF receptor may provide an appropriate model to study various PAF-related lung diseases, including bronchial asthma. Acknowledgment : The writers wish to thank Ms. I. Suganuma and Dr. H. Sasai for their technical assistance, and Drs. T. Izumi, K. Kume, E. Sudo, T. Aoki, and T. Oka for their helpful suggestions and assistance.

References 1. Prescott, S. M., G. A. Zimmerman, and T. M. McIntyre. 1990. Plateletactivating factor. J. Biol. Chem. 265:17381–17384. 2. Chao, W., and M. S. Olson. 1993. Platelet-activating factor: receptors and signal transduction. Biochem. J. 292:617–629. 3. Izumi, T., and T. Shimizu. 1995. Platelet-activating factor receptor: gene expression and signal transduction. Biochim. Biophys. Acta 1259:317– 333. 4. Honda, Z.-i., M. Nakamura, I. Miki, M. Minami, T. Watanabe, Y. Seyama, H. Okado, H. Toh, K. Ito, T. Miyamoto, and T. Shimizu. 1991. Cloning by functional expression of platelet-activating factor receptor from guinea-pig lung. Nature 349:342–346. 5. Nakamura, M., Z.-i. Honda, T. Izumi, C. Sakanaka, H. Mutoh, M. Minami, H. Bito, Y. Seyama, T. Matsumoto, M. Noma, and T. Shimizu. 1991. Molecular cloning and expression of platelet-activating factor receptor from human leukocytes. J. Biol. Chem. 266:20400–20405. 6. Ye, R. D., E. R. Prossnitz, A. H. Zou, and C. G. Cochrane. 1991. Characterization of a human cDNA that encodes a functional receptor for platelet activating factor. Biochem. Biophys. Res. Commun. 180:105– 111. 7. Kunz, D., N. P. Gerard, and C. Gerard. 1992. The human leukocyte platelet-activating factor receptor. cDNA cloning, cell surface expression, and construction of a novel epitope-bearing analog. J. Biol. Chem. 267: 9101–9106. 8. Sugimoto, T., H. Tsuchimochi, C. G. McGregor, H. Mutoh, T. Shimizu, and Y. Kurachi. 1992. Molecular cloning and characterization of the platelet-activating factor receptor gene expressed in the human heart. Biochem. Biophys. Res. Commun. 189:617–624. 9. Bito, H., Z.-i. Honda, M. Nakamura, and T. Shimizu. 1994. Cloning, expression and tissue distribution of rat platelet-activating-factor-receptor cDNA. Eur. J. Biochem. 221:211–218. 10. Ishii, S., Y. Matsuda, M. Nakamura, I. Waga, K. Kume, T. Izumi, and T. Shimizu. 1996. A murine platelet-activating factor receptor gene: cloning, chromosomal localization and up-regulation of expression by lipopolysaccharide in peritoneal resident macrophages. Biochem. J. 314: 671–678. 11. Ishii, S., T. Nagase, F. Tashiro, K. Ikuta, S. Sato, I. Waga, K. Kume, J. Miyazaki, and T. Shimizu. 1997. Bronchial hyperreactivity, increased endotoxin lethality and melanocytic tumorigenesis in transgenic mice overexpressing platelet-activating factor receptor. EMBO J. 16:133– 142. 12. Cuss, F. M., C. M. Dixon, and P. J. Barnes. 1986. Effects of inhaled platelet activating factor on pulmonary function and bronchial responsiveness in man. Lancet 2:189–192. 13. Page, C. P. 1992. Mechanism of hyperresponsiveness: platelet-activating factor. Am. Rev. Respir. Dis. 145:S31–S33. 14. Shirasaki, H., M. Nishikawa, I. M. Adcock, J. C. Mak, T. Sakamoto, T. Shimizu, and P. J. Barnes. 1994. Expression of platelet-activating fac-

Nagase, Ishii, Katayama, et al.: Airway Responsiveness in Transgenic Mice Overexpressing PAF Receptor

15.

16.

17.

18.

19.

20. 21.

22.

23.

24.

tor receptor mRNA in human and guinea pig lung. Am. J. Respir. Cell Mol. Biol. 10:533–537. Stafforini, D. M., K. Satoh, D. L. Atkinson, L. W. Tjoelker, C. E. Eberhardt, H. Yoshida, T. Imaizumi, S. Takamatsu, G. A. Zimmerman, T. M. McIntyre, P. W. Gray, and S. M. Prescott. 1996. Platelet-activating factor acetylhydrolase deficiency: a missense mutation near the active site of an anti-inflammatory phospholipase. J. Clin. Invest. 97:2784– 2791. Miwa, M., T. Miyake, T. Yamanaka, J. Sugatani, Y. Suzuki, S. Sakata, Y. Araki, and M. Matsumoto. 1988. Characterization of serum plateletactivating factor (PAF) acetylhydrolase: correlation between deficiency of serum PAF acetylhydrolase and respiratory symptoms in asthmatic children. J. Clin. Invest. 82:1983–1991. Rubin, A.-H. E., L. J. Smith, and R. Patterson. 1987. The bronchoconstrictor properties of platelet-activating factor in humans. Am. Rev. Respir. Dis. 136:1145–1151. Nagase, T., Y. Fukuchi, T. Matsuse, E. Sudo, H. Matsui, and H. Orimo. 1995. Antagonism of ICAM-1 attenuates airway and tissue responses to antigen in sensitized rats. Am. J. Respir. Crit. Care Med. 151:1244– 1249. Nagase, T., H. Matsui, T. Aoki, Y. Ouchi, and Y. Fukuchi. 1996. Lung tissue behaviour in the mouse during constriction induced by methacholine and endothelin-1. J. Appl. Physiol. 81:2373–2378. Mead, J. 1961. Mechanical properties of lungs. Physiol. Rev. 41:281–330. James, A. L., J. C. Hogg, L. A. Dunn, and P. D. Paré. 1988. The use of the internal perimeter to compare airway size and to calculate smooth muscle shortening. Am. Rev. Respir. Dis. 138:136–139. Martin, T. R., N. P. Gerard, S. J. Galli, and J. M. Drazen. 1988. Pulmonary responses to bronchoconstrictor agonists in the mouse. J. Appl. Physiol. 64:2318–2323. Chen, X.-S., J. R. Sheller, E. N. Johnson, and C. D. Funk. 1994. Role of leukotrienes revealed by targeted disruption of the 5-lipoxygenase gene. Nature 372:179–182. Tokuyama, K., J. O. Lötvall, P. J. Barnes, and K. F. Chung. 1991. Mechanism of airway narrowing caused by inhaled platelet-activating factor: role of airway microvascular leakage. Am. Rev. Respir. Dis. 143:1345– 1349.

1627

25. Stewert, A. G., P. N. Dubbin, T. Harris, and G. J. Dusting. 1990. Platelet-activating factor may act as a second messenger in the release of icosanoids and superoxide anions from leukocytes and endothelial cells. Proc. Natl. Acad. Sci. U.S.A. 87:3215–3219. 26. Goulet, J. L., J. N. Snouwaert, A. M. Latour, T. M. Coffman, and B. H. Koller. 1994. Altered inflammatory responses in leukotriene-deficient mice. Proc. Natl. Acad. Sci. U.S.A. 91:12852–12856. 27. O’Byrne, P. M., G. D. Leikauf, H. Aizawa, R. A. Bethel, I. F. Ueki, M. J. Holtzman, and J. A. Nadel. 1985. Leukotriene B 4 induces airway hyperresponsiveness in dogs. J. Appl. Physiol. 59:1941–1946. 28. Mazzoni, L., J. Morley, C. P. Page, and S. Sanjar. 1985. Introduction of airway hyperreactivity by platelet activating factor in the guinea-pig. J. Physiol. (Lond.) 365:107. 29. Hozawa, S., Y. Haruta, S. Ishioka, and M. Yamakido. 1995. Effects of a PAF antagonist, Y-24180, on bronchial hyperresponsiveness in patients with asthma. Am. J. Respir. Crit. Care Med. 152:1198–1202. 30. Wang, C. G., T. Du, L. J. Xu, and J. G. Martin. 1993. Role of leukotriene D4 in allergen-induced increases in airway smooth muscle in the rat. Am. Rev. Respir. Dis. 148:413–417. 31. Sandford, A., T. Weir, and P. Paré. 1996. The genetics of asthma. Am. J. Respir. Crit. Care Med. 153:1749–1765. 32. DeSanctis, G. T., M. Merchant, D. R. Beier, R. D. Dredge, J. K. Grobholz, T. R. Martin, E. S. Lander, and J. M. Drazen. 1995. Quantitative locus analysis of airway hyperresponsiveness in A/J and C57BL/6J mice. Nat. Genet. 11:150–154. 33. Mutoh, H., H. Bito, M. Minami, M. Nakamura, Z. Honda, T. Izumi, R. Nakata, Y. Kurachi, A. Terano, and T. Shimizu. 1993. Two different promoters direct expression of two distinct forms of mRNAs of human platelet-activating factor receptor. FEBS Lett. 322:129–134. 34. Hattori, M., H. Adachi, M. Tsujimoto, H. Arai, and K. Inoue. 1994. Miller-Dieker lissencephalopathy gene encodes a subunit of brain platelet-activating factor acetylhydrolase. Nature 370:216–218. 35. Tjoelker, L. W., C. Wilder, C. Eberhardt, D. M. Stafforini, G. Dietsch, B. Schimpf, S. Hooper, H. L. Trong, L. S. Cousens, G. A. Zimmerman, Y. Yamada, T. M. McIntyre, S. M. Prescott, and P. W. Gray. 1995. Anti-inflammatory properties of a platelet-activating factor acetylhydrolase. Nature 374:549–553.