Mechanism of Airway Narrowing Caused by Inhaled Platelet-activating Factor Role of Airway Microvascular LeakageH

KENICHI TOKUYAMA, JAN O. LOTVALL, PETER J. BARNES, and K. FAN CHUNG Introduction

PAF is a potent mediator of airway inflammation, and it can mimic severalfeatures observed in the asthmatic airways (1).Thus, PAF induces airway microvascular leakage (2), neutrophil and eosinophil chemotaxis (3), bronchoconstriction, and a prolonged increase in airway hyperresponsiveness (4-8). Inhalation of PAP causes a rapid onset of bronchoconstriction in several species including humans (5, 6) and guinea pigs (7, 8). However, the underlying mechanism of the response remains to be elucidated. In vitro human bronchi as well as tracheal smooth muscle from guinea pigs (9) do not respond to PAF except in the presence of platelets (10). The bronchoconstrictor effect of intravenously administered PAF also depends on circulating platelets (11), suggesting that PAF causes bronchoconstriction via the release of spasmogens from platelets such as histamine, 5HT, or thromboxane. However, the effect of inhaled PAF is not inhibited by platelet depletion in guinea pigs (7). In addition, prostacyclin infused in amounts sufficient to inhibit ex vivo PAF-induced platelet aggregation does not inhibit PAF-induced bronchoconstriction in humans (12). In contrast to other spasmogens, there is no relationship between the responsiveness to the inhalation of PAF and that to cholinergic agents in humans (5, 13).Further, no difference is found in the responsiveness to PAF between normal and asthmatic subjects (6, 13). Beta-adrenergic agonists, in an inhaled dose that completely blocks the bronchoconstrictor response to an inhaled methacholine, only partially protects against the PAF-induced airway response (13). Similarly in guinea pigs in vivo PAF-induced airway narrowing is only poorly reversed by a beta-agonist compared with bronchoconstriction induced by a peptide bronchoconstrictor bombesin (14). A possible explanation

SUMMARY In order to study the mechanism of airway narrowing after Inhaled platelet-activating factor (PAF) we measured concomitant changes In lung resistance (RL)and In airway microvascular leakage In anesthetized guinea pigs. RL and Its recovery after hyperinflation at 5 min were measured until 6 min after PAF aerosol (0.1, 0.3, 1, and 3 mM), and In the case of 3 mM PAF alao until 10 min. Microvascular leakage In trachea, main bronchi, and proximal and distal Intrapulmonary airways was determined by measurement of extravaseted Evans blue dye content. For comparison, the responses to Inhaled histamine (3 mM) and 5-hydroxytryptamlne (5HT) (3 mM), which act directly on airway smooth muscle, ware also examined. Inhaled PAF Increased RL dose-depandently, with a maximal response (peak RL) at 4 min after the Inhalation, whereas the response to histamine or 5HT was maximal within a taw seconds after the Inhalation. Peak RL (cm H20/ml/s) was significantly less after PAF (1.03 ± 0.09) than after histamine (8.39 ± 1.07) or 5HT (18.3 ± 6.48), although there was no significant dlffarence In RL after hyperinflation (recovary RL). No additional Increase In RL was seen betwaen 5 and 10 min after exposure. PAF caused a dose-dependent Increase In Evans blue dye extravasation; 3 mM PAF Induced significantly higher leakage than did histamine or 5HT at all airway levals at 6 min. PAF did not cause any additional extravasation of Evans blue dye at 10 min compared with that at 6 min after exposure. After PAF,recovery RL, as wall as peak RL, correlated significantly with the degree of Evans blue dye extravasetlon at all airway levals. PAFwas comparably more potent than histamine and 5HT In Inducing airway microvascular leakage than In causing Increase In RL. These dsta suggest that airway edema resulting from airway microvascular leakage Is an Important component of airway narrowing Induced by Inhalation of PAF. AM REV RESPIR DIS 1991: 143:1345-1349

for these results is that at least part of the acute airway narrowing observed after inhaled PAF may be due to mechanisms in addition to airway smooth muscle contraction such as airway edema resulting from an increase in airway microvascular permeability (2). The purpose of the present study was firstly to . elucidate whether inhaled PAP can induce airway microvascular leakage, as has been shown for intravenously administered PAF, and secondly toevaluate the role of microvascular leakage for the airway narrowing induced by inhaled PAP.Wetherefore studied the time course of the acute changes in lung resistance and microvascular leakage after PAF inhalation in the same animal. The effects of inhaled histamine or 5-hydroxytryptamine (5HT), which havedirect contractile effects on airway smooth muscle (15), were also studied. Methods We studied pathogen-free Dunkin-Hartley guinea pigs weighing 350 to 600 g. Animals

were anesthetized with an initial dose of urethane (6 to 8 ml/kg; 250/0 wt/vol in 0.9% saline) injected intraperitoneally. Additional urethane was given as required to maintain anesthesia. A tracheal cannula (10 mm in length with a 2.7-mm inner diameter) was inserted into the lumen of the cervical trachea through a tracheostomy and secured with a suture. A polyethylene catheter was inserted into the left carotid artery to monitor blood pressure and heart rate with a pressure trans-

(Received in originalform February 14, 1990 and in revised form December 6, 1990) 1 From the Department of Thoracic Medicine, National Heart & Lung Institute, and Brompton Hospital, London, United Kingdon. 2 Supported by the Medical Research Council and by the Clinical Research Committee of the Royal Brompton and National Heart Hospitals, United Kingdom; and by the Respiratory Research Group, Draco, Lund, Sweden. . 3 Correspondence and requests for reprints should be addressed to Dr. K. F. Chung, Department ofThoracic Medicine, National Heart & Lung Institute, Dovehouse Street, London, SW3 6LY, UK.

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nebulized for 30 breaths. An additional 5 breaths were given through the nebulizer circuit to clear the system of aerosol. RL, transpulmonary pressure, and mean blood presMeasurements of Airway Function sure wererecorded everyminute. Five minutes Guinea pigs were placed in a supine position after inhalation of vehicleor drug, the animals were hyperinflated with twice the tidal volwith the intratracheal cannula connected to a constant-volume mechanical ventilator ume by manually blocking the outflow of the ventilator, and 1 min later they were discon(Model 50-1718; Harvard Apparatus Ltd, Edenbridge, UK). A tidal volume of 10ml/kg . nected from the ventilator. In two different and a frequency of 60 breaths/min were used. groups of animals, Evans blue dye content Transpulmonary pressure was measured with in airway tissues was measured immediately a pressure transducer (Model FCO 40; ± or 2 min after the inhalation of 3 mM PAF 1,000 mm H 2 0 ; Furness Controls Ltd, Bex- in order to study the duration of microvascular leakage induced by inhaled PAF. hill, UK), with one side attached to a catheter inserted into the right pleural cavity and the other side attached to a catheter connectDetermination of Plasma Extravasation ed to a side port of the intratracheal cannula. After disconnection from the ventilator the The ventilatory circuit had a total volume of 20 ml. Airflow was measured with a pneu- thoracic cavity was opened, and a cannula motachograph (Model FIL; Mercury, Glas- was inserted into the aorta through the left gow, UK) connected to a transducer (Model ventricle. Animals were perfused with 0.9% FCO 40; ± 20 mm H 20 ; Furness Controls). NaCI solution at a pressure of 100 mm Hg All signals were recorded on a six-channel to remove intravascular dye. The lungs were recorder (Model MX6; Lectromed ux, Ltd, removed, and the parenchyma of tissue was Hertfordshire, UK). Lung resistance (RL)was carefully scraped off. The trachea, main broncalculated using the method of von Neergaard chi, and intrapulmonary airways were separated from each other, and the intrapulmoand Wirz (16). Aerosols were generated with an ultrason- nary airways were divided lengthwise into two ic nebulizer (Pulmosonic Model 2511; DeVil- equal portions, arbitrarily named proximal biss Co., Somerset, PA), and were ad- (P-IPA) and distal (D-IPA). All tissues were ministered to the airways through a separate weighed wet. Evans blue dye was extracted 0 ventilator system that bypassed the pneu- in 2 ml of formamide at 40 C for 24 hand motachograph. The volume of this circuit was measured in a spectrophotometer (Model 50 ml. The output from the nebulizer at the 8480; Phillips, Cambridge, UK) at 620 nm, port of the tracheal cannula, measured with The extracted Evans blue dye was quantified a tidal volume of 5 ml and a frequency of by interpolation on standard curve of dye con60 breaths/min, was 35 Ill/min. The mean centrations in the range of 0.5 to 10 ug/ml particle size produced by the nebulizer was and expressed as ng dye/mg tissue. The Evans 3.8 11m, with a geometric standard deviation blue dye measurement has been previously of 1.3, as measured with a laser droplet and shown to highly correlate with the extravasaparticle analyzer (Model 2600c; Malvern In- tion of radiolabeled albumin in guinea pig airways (17). struments, Worcester, UK). ducer. The right external jugular vein was cannulated for the administration of Evans blue dye.

Protocol Animals were divided into eight groups in order to compare the effects of inhaled PAF (0.1, 0.3, 1, and 3 mM), histamine (3 mM), or 5HT (3 mM) on airway microvascular leakage and increases in lung resistance up to 6 min after exposure. In an additional group, the effects of PAF (3 mM) up to 10 min were observed. Wealso studied the effects of a specific PAF-receptor antagonist (WEB 2086; 50 and 500 ug/kg given intravenously) on the responses to PAF (3 mM). As a control challenge, we used 0.9070 saline, which was the diluent of histamine or 5HT. PAF was stored in 100% ethanol at 20 0 C at a concentration of 10mg/ml. On the study day, it was diluted with 0.9% saline containing 0.25% bovine serum albumin (BSA). Therefore, we also examined the response to 15% ethanol in 0.9% saline containing 0.21% BSA (the same composition of the diluent of the 3-mM PAF solution) in control studies. Ten minutes after connection to the ventilator, Evans blue dye (20 mg/kg) was administered intravenously. One minute later, the nebulizer circuit was opened, and each mediator or its vehicle was

Drugs and Chemicals The following drugs and chemicals wereused: urethane, histamine chloride, 5-hydroxytryptamine, and bovine serum albumin (Sigma Chemical Co Ltd, Poole, UK); PAF (1-o-hexadecyl-2-acetyl-sn-glycero-3-phosphorylcholine) (Bachem Feinchemikalien AG, Budendorf, Switzerland); ethanol (BDH Chemicals, Poole, UK); 0.9% wt/vol sodium chloride in distilled water (Travenol Laboratories, Thetford, UK). Data Analysis Data are reported as means ± SEM. Nonparametric analysis of variance (KruskalWallismethod) was used to determine the significant variance between groups. If a significant variance was found, a Mann-Whitney U test was used to test for significant difference betweenindividual groups. For correlation between variables, Spearman's rank analysis Was used. A p value less than 0.05 was considered significant. Data wereanalyzed by a computer using standard statistical packages.

Results

Time Course of Lung Resistance There was no significant difference in baseline RL between any of the groups studied. The response to vehicle for PAF (15070 ethanol in 0.9070 NaCI containing 0.21070 BSA) was not significantly different from that of 0.9070 NaCI alone, which was the diluent of histamine or 5HT. Inhalation of PAF caused significant increase in RL. The mean RLwas maximal approximately 4 min after inhalation of PAF (figure 1, left panel). The maximal RL (peak RL) was 0.69 ± 0.13 (n = 4), 0.67 ± 0.09 (n = 4), 1.12 ± 0.10 (n = 4), and 1.03 ± 0.09 em H 20/mIls (n = 6) after 0.1,0.3, 1, and 3 mM PAP, respectively, and 0.45 ± 0.04 (n = 6) and 0.49 ± 0.03 em H 20/mIls (n = 4) after saline and vehicle for PAF, respectively. Peak RL was significantly higher after 0.3, 1, and 3 mM PAF than after 0.9070 NaCI (p < 0.05, p < 0.05, and p < 0.01, respectively). Recoveryof RL after hyperinflation after 1 and 3 mM PAF (to 0.78 ± 0.12and to 0.75 ± 0.06 em H 20/mIls, respectively) was also significantly less than after 0.9070 NaCI (to 0.35 ± 0.03 cm H 20/mIls). Histamine or 5HT (3 mM) caused a marked increase in RL, with a maximal response observed immediately after inhalation (8.39 ± 1.07 and 18.3 ± 6.48 em H 20/mIls, respectively). These values were significantly higher than those after 3 mM PAF (p < 0.01), as well as after 0.90/0 NaCI (p < 0.01). The time course of RLafter 3 mM histamine, 5HT, and PAF and after 0.9% NaCI is shown in figure 1, right panel. Recoveryof RLwas also significantly less after histamine or 5HT (to 1.23 ± 0.26 and to 0.94 ± 0.21 em H 20/mIls, respectively)compared with 0.9070 NaCI (to 0.35 ± 0.03 em H 20/mIls, p < 0.01), but not compared with PAF (to 0.76 ± 0.09 em H 20/mIls). In a separate group of animals, there was no additional increase in RLinduced by PAF between 5 and 9 min, and at 10min, recovery RLafter PAF was 0.59 ± 0.12 em H 20/mIls. There was no significant decrease in mean blood pressure after inhalation of PAP or 5HT compared with sham stimulation, although it fell by 34.1 ± 3.6% after histamine (p < 0.01).

Extravasation of Evans Blue Dye At all airway levels, there was no significant difference in extravasation of Evans blue dye after inhaled 0.9% NaCI or vehicle for PAP. Inhaled PAF dose-dependently increased the amount of extravasated dye. The minimum concentrations

AlffWAY NARROWING CAUSED BY INHALED PLATELET-ACTlVATING FACTOR

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Mechanism of airway narrowing caused by inhaled platelet-activating factor. Role of airway microvascular leakage.

In order to study the mechanism of airway narrowing after inhaled platelet-activating factor (PAF) we measured concomitant changes in lung resistance ...
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