INTRODUCTION

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Platelet-activating factor (PAF) is a potent ether-linked phospholipid mediator of inflammation that is considered to have a role in the pathogenesis of bronchial asthma and other pulmonary disorders (1, 2). We have shown that PAF induced or worsened gas exchange abnormalities in normal subjects (3) and in patients with mild asthma (4). These disturbances were characterized by an increased dispersion of pulmonary blood flow, including the development of low ventilation-perfusion (A/) areas, identical to the entire spectrum of A/ inequalities seen in patients with bronchial asthma (5). Although A/ mis matching in patients with asthma is akin to airway narrowing by both inflammation and bronchoconstriction, the precise mechanism by which A/ inequalities may occur still remains elusive. We suggested that the A/ defects could be related to an increased tracheobronchial vascular permeability induced by PAF, therefore supporting the notion that PAF may play a key role as a putative mediator of inflammation in airways (3, 4).

Previous studies in asthmatic patients have consistently shown a poor correlation between the behavior of reduced maximal expiratory airflow rates and abnormal pulmonary gas exchange, namely, arterial blood gases and their major intrapulmonary determinant, A/ imbalance, in individual patients and also within clinically similar asthma patients' category, such that it can be extended across the full constellation of asthma severity (5, 6). Conceivably, these intriguing findings reflect two different pathophysiologic phenomena and concur with the hypothesis that decreased spirometric indices r eflect reduction of airway caliber in larger and middle-size bronchi, whereas pulmonary gas exchange disturbances predominantly refer to structural changes in distal small airways (6). Thus, the latter changes could be more preferentially related to airway inflammation rather than to airflow obstruction by itself. Notwithstanding, cause and effect relationship will be very difficult to establish in humans.

Salbutamol, a short-acting beta-adrenergic agonist, inhibits PAF-induced increased airway resistance and the systemic (cough, facial flushing, and feeling of warmth), cellular (peripheral blood neutropenia), and gas exchange (impaired arterial oxygenation and A/ imbalance) effects in normal subjects (7, 8). We postulate that these effects of salbutamol could be related preferentially to an inhibition of PAF-induced precapillary and postcapillary endothelial constriction in the bronchial microcirculation (9, 10), although its potent relaxant effect on airway smooth muscle cannot be overlooked. If so, an anticholinergic agent devoid of vascular effects such as ipratropium bromide should prevent PAF-induced bronchoconstriction but not interfere with its systemic, cellular, and gas exchange effects. The present study was undertaken to test this hypothesis by assessing the cellular, lung mechanical, and gas exchange responses to PAF after ipratropium bromide (80 µg) and salbutamol (300 µg) given by inhalation in patients with mild asthma.

	METHODS

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Patients

Eight nonsmokers with mild asthma were recruited for the study (Table 1), which was approved by the Ethical Committee of Hospital Clínic. All subjects gave informed written consent after the purpose, risks, and potential benefits of the study were explained to them. Inclusion criteria were: no respiratory infection or exacerbation of asthma within the preceding 6 wk; FEV₁  70% predicted and positive methacholine bronchial challenge (PD₂₀ < 4.0 µmol); maintenance therapy with aerosol short-acting beta-adrenergics and/or inhaled corticosteroids, but no previous treatment with oral steroids; absence of any systemic or cardiopulmonary disease other than asthma.

Measurements

Blood samples were collected anaerobically through a catheter inserted into the radial artery. Total white cell counts in arterial blood were measured with a Technicon H.1^TM System (Technicon, Tarrytown, NY). Arterial PO₂, PCO₂, and pH were analyzed in duplicate using standard electrodes (IL 1302; Instrumentation Laboratories, Milano, Italy). Hemoglobin concentration was measured by a Co-oximeter (IL 482; Instrumentation Laboratories). Oxygen uptake (O₂) and CO₂ production (CO₂) were calculated from mixed expired O₂ and CO₂ concentrations measured by mass spectrometry (Multigas Monitor MS2; BOC-Medishield, London, UK). Minute ventilation (E) and respiratory rate (RR) were measured using a calibrated Wright spirometer (Respirometer MK8; BOC-Medical, Essex, UK). The AaPO₂ was calculated according to the alveolar gas equation using the measured respiratory exchange ratio (R). The multiple inert gas elimination technique (MIGET) estimated the distributions of A/ ratios without sampling mixed venous inert gases in the customary manner, a modality that can be used with similar accuracy (12) in all but one patient. With this approach cardiac output needs to be directly measured by dye dilution technique (DC-410; Waters Instruments Inc., Rochester, MN) using a 5-mg bolus of indocyanine green injected through a catheter placed percutaneously in a vein of the arm while mixed venous inert gas concentrations are computed from mass balance equations (12). The duplicate samples of each set of measurements were treated separately, the final data resulting in the average of variables determined from both A/ distributions at each point in time. Maintenance of steady-state conditions after PAF challenge was demonstrated by stability ( ± 5%) of both ventilatory and hemodynamic variables, and by the close agreement between duplicate measurements of mixed expired and arterial O₂ and CO₂ (within ± 5%). These conditions were met in all patients throughout the whole period of study.

Total resistance of the respiratory system (Rrs) was measured by the forced oscillation technique and its analysis restricted to 8 Hz (3, 4). A three-lead electrocardiogram, heart rate (HR), and systemic pressure (Ps) were continuously recorded throughout the whole study (HP 7830A Monitor and HP 7754B Recorder; Hewlett-Packard, Waltham, MA).

Study Design

A randomized double-blind crossover design was used to compare the effect of salbutamol with that of ipratropium bromide on PAF- induced effects, with subjects breathing room air and seated in an armchair. Medication was withheld for 12 h before arrival to the laboratory. Once the inert gas solution had been infused for at least 45 min to allow for the establishment of adequate steady-state conditions, baseline measurements were performed. All subjects were challenged on two occasions 1 wk apart with inhaled PAF 30 min after the administration of either ipratropium bromide (two puffs = 80 µg) or salbutamol (three puffs = 300 µg), using a regular metered-dose inhaler with an approximately 1-L holding chamber, one puff at a time, and a set of measurements was taken 15 min later. It has been shown that 80% of the maximal bronchodilation produced by ipratropium bromide can be achieved with a cumulative dose of 72 µg (13). Patients were challenged with PAF (C₁₆) (1-0-Hexadecyl-2-acetyl-sn-glycero-3-phosphocholine) (18 µg) (Novabiochem AG, Lucerne, Switzerland). Duplicate measurements were taken at 5, 15, and 45 min after PAF inhalation, as described previously (6). All sets of measurements consisted of the following steps in sequence: inert gas sampling and ventilatory recordings; respiratory gas sampling; hemodynamic measurements; sampling for circulating white blood cells; measurements of Rrs.

Statistics

Results are expressed as mean ± SE. Changes in neutrophils, Rrs, arterial blood gases, and A/ inequalities were assessed by an analysis of variance (ANOVA) model appropriate to the two-period two-treatment crossover design, to determine the effect of ipratropium bromide compared with that of salbutamol, hence allowing for intraindividual comparisons at each time point. Homoscedasticity was obtained by logarithmic transformation. This statistical approach was identical to that used in our previous studies (7, 8). Significance was set at p  0.05 in all instances.

	RESULTS

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Baseline Data and Effects of Inhaled Ipratropium and Salbutamol before PAF

Baseline measurements for all patients were similar to those reported in our previous investigation (7), without differences between ipratropium bromide and salbutamol studies (Tables 1 and 2 and Figure 1). Compared with ipratropium bromide, however, salbutamol produced an increase of T (from 6.2 ± 0.4 to 6.9 ± 0.4 L/min) (p < 0.02) before PAF inhalation, whereas the first moment of the A/ distribution (the mean A/ ratio of the perfusion distribution, ) decreased (from 0.78 ± 0.06 to 0.70 ± 0.05) (p < 0.03), an effect also shown previously in normal subjects after they had received salbutamol (7).

View larger version (23K): [in this window] [in a new window]   Figure 1.   Mean ± SE values showing peripheral neutrophils, resistance of the respiratory system, alveolar-arterial PO2 difference, and ventilation-perfusion mismatching (expressed as DISP R-E*, dimensionless [14]) after PAF challenge in the two groups. Closed symbols = pretreated with ipratropium bromide (IB); open symbols = pretreated with salbutamol (s) at baseline (BL), at 15 min after bronchodilators, and at 5, 15, and 45 min. Asterisks denote significance (see RESULTS for p values).

Effects of Ipratropium Bromide and Salbutamol after PAF

Compared with pretreatment with salbutamol, after ipratropium bromide there were no significant differences in Rrs after PAF challenge, indicating that both agents were efficacious in blocking the expected PAF-induced increase of Rrs (Table 2 and Figures 1 and 2). However, after pretreatment with ipratropium bromide six subjects noticed facial flushing, and five coughed immediately; by contrast, after salbutamol only one patient had facial flushing. On the other hand, after pretreatment with ipratropium bromide there was significant neutropenia (from 3.74 ± 0.35 to 1.67 ± 0.39 × 10⁹/L) (p < 0.03) at 5 min; in addition, Pa_O2 showed a trend to decrease (from 101 ± 4 to 87 ± 5 mm Hg) (p = 0.08) at 5 min, which persisted at 15 min (to 93 ± 5 mm Hg) (p = 0.05), whereas AaPO₂ increased markedly (from 13 ± 3 to 30 ± 5 mm Hg and to 24 ± 3 mm Hg) at 5 and 15 min, respectively (p < 0.02 each), returning to baseline values at 45 min. These findings were paralleled by a considerable A/ deterioration, essentially illustrated by a marked increase of the dispersion of pulmonary blood flow (log SDQ) (from 0.57 ± 0.09 to 0.87 ± 0.11 and 0.73 ± 0.10) at 5 and 15 min, respectively (p < 0.04 each) along with an increase of an overall index of A/ inequality (DISP R-E*) (the combined dispersion of both blood flow and ventilation distributions corrected for dead space [14]) (from 3.46 ± 0.80 to 6.35 ± 1.36 and 4.91 ± 1.09) at 5 and 15 min, respectively (p < 0.05 each), to return to baseline values at 45 min. Individually, all patients pretreated with ipratropium bromide had a deterioration in pulmonary gas exchange after PAF, whereas in all but one patient, PAF-induced gas exchange defects were prevented after salbutamol (Figure 2). Similarly, the first moment of the A/ distributions (the mean A/ ratio of the ventilation distribution, ) increased (from 1.28 ± 0.12 to 1.42 ± 0.08) at 15 min (p < 0.02) after ipratropium bromide.

View larger version (24K): [in this window] [in a new window]   Figure 2.   Individual time courses of alveolar-arterial PO2 and ventilation-perfusion inequalities (expressed as DISP R-E*, dimensionless [14]) (n = 7) after inhaled PAF with pretreatment with inhaled ipratropium bromide or with salbutamol at baseline (BL), at 15 min after bronchodilators, and at 5, 15, and 45 min (see Table 1 and Figure 1 for other abbreviations).

Overall, the changes observed in neutrophils and both respiratory and inert gas exchange descriptors after ipratropium bromide were similar to those previously detected in unpremedicated patients with asthma (4), or in normal subjects unpretreated (3) or pretreated with saline (vehicle) (7, 8), indicating that ipratropium bromide had no effect on the PAF-induced abnormalities. By contrast, salbutamol prevented all PAF-induced functional defects, including systemic and neutrophil changes. Ventilatory and hemodynamic variables and all the other gas exchange indices remained unchanged between studies after PAF challenge.

	DISCUSSION

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The unique finding of this study is that, in patients with mild asthma, ipratropium bromide administered at a maximal bronchodilating dosage had a protective effect on bronchoconstriction but not on the systemic, neutropenic, and pulmonary gas exchange responses provoked by PAF challenge. By contrast, salbutamol prevented all PAF-induced lung function disturbances, including facial flushing and peripheral neutropenia. Our findings complement and extend our previous investigations in normal subjects that pretreatment with inhaled salbutamol suppressed all PAF-induced effects (3, 7, 8). It is of note that a lower dose of salbutamol (200 µg) had no effect on the flushing, neutropenia, and bronchoconstriction observed after a higher dosage of inhaled PAF in normal subjects (15).

The A/ mismatching, expressed as an increase of the dispersion of pulmonary blood flow (log SDQ), observed in the current study in the asthmatic patients premedicated with ipratropium bromide was quantitatively similar to that shown in healthy subjects (3, 7) in whom a higher dose (24 µg) of inhaled PAF was used and to that in patients with mild asthma inhaling a lower dose (12 µg) of PAF (4); likewise, the falls in peripheral blood neutrophil counts were of a similar order of magnitude to those of normal subjects (3, 8) or even greater than in asthmatics (4). In the current study and in the previous one (4), in patients with asthma, however, inhaled PAF was qualitatively detrimental to pulmonary gas exchange, provoking A/ defects in a pattern similar to that commonly observed in patients with moderate to severe asthma (5). In the present study, the deterioration of A/ relationships resulted mainly from an increase in the dispersion of pulmonary blood flow caused by the development of poorly ventilated A/ units, akin to the underlying pathophysiology of bronchial asthma (5). Our data are, however, at variance with those obtained by Smith and coworkers (16) in normal subjects and in asthmatics, in whom pretreatment with atropine paradoxically enhanced PAF-induced bronchoconstriction. Collectively, these A/ findings after salbutamol and ipratropium bromide strengthen the view that bronchoconstriction and gas exchange disturbances in patients with asthma are related to two different pathophysiologic components. Thus, a bronchodilator acting predominantly on larger airways and devoid of other effects such as ipratropium bromide prevented PAF-induced increased resistance of the respiratory system only without influencing A/ deterioration or the neutropenic and systemic responses. By contrast, salbutamol, a bronchodilator with potent vasodilator effects, blocked all PAF-induced effects, possibly by modulating abnormal vascular permeability-increasing mediators that operate directly on the venular endothelium. In this respect, gas exchange measurements can emerge as a better tool than any other lung function test to more accurately identify the pathobiologic events that involve more peripheral quiet regions of the lungs.

In our previous work, in both healthy subjects (3, 7, 8) and in patients with mild asthma (4), we suggested that pulmonary gas exchange abnormalities and the simultaneous modest increase of Rrs caused by inhaled PAF were more related to narrowing of airway caliber secondary to increased microvascular leakage than to a primary reversible constrictor effect (3). Platelet-activating factor, like other putative inflammatory mediators in the lungs, induces increased vascular recruitment and/or vascular engorgement, vasodilation, and increased vascular permeability, thereby causing exudation of protein-rich plasma around (in mucosa, submucosa, and/or adventitia) and within the airway lumen (17, 18). It has been suggested that abnormal airway microvascular leakage can magnify the bronchoconstrictor response by several mechanisms such as increasing mucosal and/or submucosal thickness, interfering with the mechanical properties of the airway wall, uncoupling of the airway from the surrounding lung parenchyma, and/or filling airway interstitial spaces, which together result in decreased airway caliber and increased resistance of the tracheobronchial tree (18). PAF may also act directly on postcapillary venular endothelial cells in the bronchial microcirculation (9, 10). Salbutamol could also prevent the ensuing release of other mediators into the pulmonary circulation with potential regional vasodilator effects that can disturb the matching of ventilation and perfusion at the alveolar level, hence antagonizing further A/ disturbances. It is of note that these potential vasodilator effects of salbutamol on the bronchial and pulmonary circulations, precluding the A/ deterioration by PAF as alluded to above, do not contend with their impact on A/ worsening in patients with asthma. We have previously shown that inhaled salbutamol (total doses, 600 and 300 µg) does not alter the underlying A/ status in patients with either severe acute (19) or persistent (20) asthma, respectively.

Likewise, the beneficial role of salbutamol in preventing PAF-induced neutrophil sequestration in the lungs may indicate an antiedema property that may result from inhibition of PAF-induced inflammation in airway wall, possibly amplified by its potent relaxant effect on conducting airways. This interpretation is consistent with the inhibition by ₂-adrenergic agonists of the increased tracheobronchial microvasculature permeability provoked by PAF (21, 22) and also by histamine (23). Indirect evidence (22) suggests, however, that the protective role of salbutamol on gas exchange may be more related to an inhibition of a PAF-induced venoconstrictor effect on the airway microcirculation (23). Moreover, salbutamol causes vasodilatation that can increase the postmicrovascular to premicrovascular resistance ratio of the bronchial circulation, thereby decreasing the hydrostatic pressure and subsequent plasma exudation (28). The reduction of hydrostatic pressure in the airway capillary network could decrease the degree of airway submucosal and adventitial swelling, thereby preventing the narrowing of the caliber in distal airways, resulting in pulmonary gas exchange abnormalities. Salbutamol would have thus enhanced the ability of endothelial cells to either minimize and/or close PAF-induced interendothelial gap junctions by facilitating their relaxation.

If pretreatment with salbutamol has a protective effect on the transient sequestration of neutrophils in the pulmonary circulation produced by PAF, it may reduce the activation of these cells in the lungs and the subsequent cascade of other released mediators that may also play a role in the PAF-induced pulmonary function abnormalities. Both facial flushing and cough induced by PAF have been attributed to the release of by-products, possibly derived from neutrophils, acting systemically (23).

Taken in sum, the inhibition of PAF-induced bronchoconstriction but not of neutropenia, systemic effects, and gas exchange disturbances by ipratropium bromide, but the inhibition of all these PAF-induced changes by salbutamol in this subset of asthmatic patients reinforces the view that beta-adrenergic agonists may block the postcapillary venoconstriction of the bronchial circulation provoked by PAF. These findings enhance the concept that PAF can be viewed as a putative mediator of inflammation in human airways, although a mechanistic relationship cannot be clearly established.