INTRODUCTION

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A dysfunctioning pulmonary surfactant is considered to be an important cause of infant and acute or adult respiratory distress syndromes (IRDS and ARDS). These disorders are characterized by atelectasis, nonhydrostatic pulmonary edema, and impaired gas exchange. A surfactant deficiency, considered to be affecting alveolar function, is widely regarded as a major factor in the pathogenesis (1). In contrast to this emphasis on alveolar function, attention has not been focused on the possible involvement of pulmonary surfactant in the pathophysiology of respiratory diseases such as asthma, which is principally affecting conducting airways. However, theoretical considerations and experimental data support the concepts that pulmonary surfactant is essential for a normal function of terminal conducting airways, and that a surfactant dysfunction may cause those airways to become temporarily blocked, thereby contributing to an increased airway resistance.

Asthma is characterized by a chronic airway inflammation (2) with an invasion of plasma proteins (3, 4), some of which, like fibrin, are potent surfactant inhibitors (5). For that reason it is conceivable that in asthma there might very well be a surfactant dysfunction. Furthermore, it has been reported that guinea pigs, sensitized with ovalbumin and then challenged with aerosolized antigen, reacted with a leakage of plasma proteins into the airway, a markedly increased airway resistance, and an altered surfactant performance, indicating a dysfunction (10). It has also been shown that prophylactic treatment of sensitized animals with intratracheal instillation of surfactant reduces the deteriorating lung function that otherwise would have developed (11). In studies from another laboratory it was demonstrated that treatment of immunized guinea pigs with aerosolized surfactant alleviates an increase in airway resistance (12). Moreover, in heterozygous SP-B-deficient mice air trapping was observed, suggesting that airway obstruction might have been due to a surfactant dysfunction caused by the SP-B deficiency (13). Recently, van de Graaf and colleagues (14) described how the BALF levels of SP-A were lowered in patients with asthma, and Kurashima and colleagues (15) reported on a pilot study indicating that patients suffering from an acute asthma attack were relieved when they inhaled aerosolized surfactant. The same investigators have reported that sputum samples from patients with asthma have a low surface activity (16). How surfactant dysfunction can develop and cause obstruction of small airways was recently reviewed (17).

Pulmonary surfactant in BALF, its surface activity, and more specifically its ability to maintain airway patency might thus be of great importance for an atopic patient with asthma, and therefore we carried out a bronchoscopy study of healthy volunteers and of patients with mild atopic asthma. We hypothesized that in an asthmatic patient, challenge with a specific antigen will induce an inflammatory reaction, which is likely to interfere with the normal function of pulmonary surfactant. We obtained BALF for baseline values from one lobe, challenged another lobe with saline solution only, and challenged a lobe in the other lung with antigen. Twenty-four hours after the challenge the bronchoscope was introduced again in order to obtain fluid from the challenged lung lobes. Consequently, we were able to compare biophysical and biochemical surfactant properties of BALF at baseline, after a challenge with vehicle only, and after a challenge with antigen.

	METHODS

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Patients

Nine healthy volunteers and 15 patients with mild asthma were enrolled for the study (Table 1). All patients had allergic asthma according to the Heart, Lung, and Blood Institute of the National Institutes of Health (18). Most of them had seasonal asthma and were tested only if they were in their symptom-free season. In response to one or more of eight common allergens (Dermatophagoides pteronyssinus, Dermatophagoides farinae, mixed grass pollen, mixed tree pollen, dog hair, feathers, cat fur, Alternaria) each patient had at least one positive skin prick test, defined as a skin wheal with a diameter > 4 mm. The allergen extract subsequently used for segmental allergen challenge was the one that had produced the largest wheal response on skin prick testing (D. pteronyssinus or grass pollen). On the day of bronchoscopy a skin wheal dose-response series was obtained with the chosen allergen. The concentration used for the endobronchial challenge was one tenth of that which elicited a skin wheal with a diameter of 3 mm. Bronchial hyperresponsiveness was determined by a modified bronchoprovocation test with histamine as described elsewhere (19). In short, histamine was nebulized into a 10-L reservoir with a pressure nebulizer (Pari Provocation test II; Starnberg, Germany). The aerosolized histamine was inhaled through a one-way valve and the concentrations were stepwise doubled. The PC₂₀ was calculated as the cumulative quantity of histamine that had reduced FEV₁ by 20% of the baseline value postinhalation of aerosolized saline solution. Current medication consisted of ₂-agonists only. None of the patients had used glucocorticosteroids, sodium chromoglycate, or theophylline for at least 6 wk.

The healthy control subjects had no history of allergic diseases or any other disorders, they had negative skin-prick tests, normal IgE levels ( 100 IU/ml), normal lung function tests, and no bronchial hyperresponsiveness (PC₂₀ > 8 mg/ml). All study subjects were nonsmokers and none of them had suffered an acute bronchitis during the 4-wk period preceding the challenge. The study was approved by the Ethics Committee of Hannover Medical School, and informed consent was obtained from each person in the study.

Segmental Allergen Challenge

Segmental allergen challenge was done as previously described (20). Prior to the procedure each patient received nebulized salbutamol (1.5 mg), atropine (0.5 mg subcutaneously), and midazolam (5 mg intravenously). Oxygen (100%) was delivered via nasal cannulae throughout the procedure (2 to 4 L/min), and oxygen saturation and heart rate were monitored with a digital oximeter. Lidocaine (4%), in a maximal volume of 3 ml was sprayed into the upper airways to achieve local anesthesia. The bronchoscope (P30; Olympus Optical, Tokyo, Japan) was then passed through the mouth or nose, and the larynx and lower airways were anesthetized with maximally 15 ml 2% lidocaine.

The instrument was wedged into the inferior lingular bronchus, which was lavaged so that baseline values could be obtained. The bronchoscope was then passed into the superior lingular bronchus, and 10 ml warm saline solution was instilled as a control challenge. Finally, the instrument was moved to the contralateral lung into the medial segment of the middle lobe where 10 ml warm allergen solution was instilled. After the saline and allergen instillations the airways were observed for 5 min, and after the bronchoscopy each subject was observed closely for 4 h, and when they left for home they were given a contact telephone number. Twenty-four hours after the bronchoscopy a subgroup of five healthy volunteers and all but one asthmatic underwent a second bronchoscopy to obtain BALF from the challenged airways. One asthmatic was not rebronchoscoped because of an asthma attack that developed after the first examination. The same premedication was used and the superior lingular bronchus and the medial middle lobe bronchus were lavaged.

Bronchoalveolar Lavage

The fiberoptic bronchoscope was wedged into the appropriate bronchus and lavage was done with warm saline solution in five 20-ml aliquots. After instillation each aliquot was aspirated with gentle suction. The BALF was pooled and the recovered volume was recorded (= recovery). Specimens of 5 ml were sent for total count of cells and for differential cell counts. The remaining fluid was filtered through sterile gauze and then centrifuged at 250 × g for 10 min to obtain a cell-free supernatant, which was stored at 28° C until further analysis. All further measurements were performed blindly.

Protein and Phospholipid Analysis

Aliquots of the cell-free supernatant were used for determination of protein concentrations according to Lowry and colleagues (21) and of phospholipid concentrations according to the method of Bartlett (22). The latter assay is based on a phosphorus determination carried out on the lipids extracted with chloroform/methanol according to Bligh and Dyer (23). All assays were performed in duplicate and the mean value was reported.

Surfactant Aggregate Separation

The cell-free supernatant was centrifuged at 48,000 × g for 60 min at 4° C to pellet large surfactant aggregates (LA). The supernatant, containing small surfactant aggregates (SA), was removed and the LA pellet was resuspended in Ringer's solution. The phospholipid contents of the LA pellets and the SA supernatants were determined as described above. By adding Ringer's solution, the phospholipid concentration of the LA suspension was adjusted to 1 mg/ml. This was in preparation for a study of surface properties with the pulsating bubble surfactometer (PBS).

Surface Activity Evaluated with the PBS

Surface activity of BALF was measured with a PBS (Electronetics, Buffalo, NY) (24) and with a capillary surfactometer (CS, see below). For the PBS, 40 µl of the LA suspension, which had been given a phospholipid concentration of 1 mg/ml, were used for filling the sample chamber with a micropipet. The surface tension used for statistical analysis of this study was the value at minimal bubble size, _min, registered after 5 min of pulsation at a rate of 20 cycles/min and at a temperature of 37° C. Before starting, bubble pulsation adsorption rate was evaluated by determining surface tension 10 s after formation of a bubble (_ads). All analog data were digitalized and recorded by computer.

Surface Activity Evaluated with the CS

Surfactant function, as it applies to the cylindrical surface of a narrow conducting airway, was evaluated with a CS. This instrument simulates the morphology and function of a terminal conducting airway with a glass capillary that in a short section is particularly narrow with an ID of 0.2 mm (25). It is in that section that liquid is likely to accumulate, but it will be prevented from doing so by a well-functioning pulmonary surfactant. A small volume (0.5 µl) of the liquid to be evaluated is deposited in this section. The lumen of the capillary will then be totally blocked but, when pressure is raised at one end of the capillary, the liquid is extruded from the narrow section. It will not return provided it contains well-functioning surfactant, but if the surfactant is at a very low concentration or functioning poorly, the liquid will return and again block the capillary lumen. Because there is a continuous flow of air through the capillary and pressure is recorded at the capillary inlet, the function of the surfactant can be evaluated. Pressure is zero if the capillary is open for free airflow, but there will be an increase in pressure when the liquid returns to block the narrow section. Pressure is recorded for 120 s, and a computer calculates the percentage of that time that the capillary is open. Well-functioning pulmonary surfactant will keep the capillary open 100%, showing an excellent ability to maintain airway patency, whereas with surfactant functioning very poorly, the value of "Open in %" will be zero.

The surfactant in the BALF was too diluted for an evaluation with the CS and had to be concentrated approximately 20 times, five times by centrifugation and four times by evaporation. A volume of 500 µl was centrifuged at 4° C and 40,000 × g for 1 h. After removal of 400 µl supernatant, the remaining 100 µl, containing the LA, was exposed to vacuum in a small test tube. Through a very fine glass capillary, dry nitrogen was sucked into the test tube in order to completely dehydrate the sample. This took 20 to 30 min, and the sample was then resuspended in 25 µl of the supernatant and carefully stirred. Five 0.5-µl aliquots were studied with the CS and the mean was reported.

Surfactant dysfunction registered with the CS is often due to inhibiting agents. To evaluate the effect of those agents the supernatant from the second centrifugation was used to dilute calf lung surfactant extract, CLSE (kindly donated by ONY Inc., Amherst, NY) from its original concentration of 35 mg/ml to 1 mg/ml. If saline solution were to be used for that dilution, the value of "Open in %" would be 100, but the presence of inhibitors in the supernatant would result in a lower percentage of openness.

Another way of examining if inhibiting agents had been the reason for the poor surfactant function seen with the CS was to remove the "inhibitors." For that reason the surfactant suspension remaining after the five assays, approximately 20 µl, was "washed." A relatively large volume of saline solution, 500 µl, was added to the sample. After careful stirring the sample was centrifuged again at 4° C and 40,000 × g for 1 h. The liquid added, 500 µl, was removed. The rest was stirred and evaluated again with the CS. Samples of the "washed" surfactant were evaluated three times.

Statistics

Results are expressed as mean ± standard error of the mean (SEM). Diversity in means of the treatment groups (baseline, saline, and allergen) within the major study groups (asthma and control subjects) were tested by Friedman's nonparametric test for paired comparison followed by Wilcoxon's rank test for individual comparison between treatment groups. Differences between the major study groups were tested by the Mann-Whitney U test (28); p values of less than 0.05 were considered significant. Post hoc Bonferroni correction was performed consistently.

	RESULTS

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Basic data and clinical characteristics of the study subjects are given in Table 1.

Cells in BALF

When the lavage was done to obtain the baseline values there was no difference between the healthy volunteers and the asthmatics with regard to recovery of BALF (78 ± 3 and 74 ± 2 ml, respectively, p = 0.16). The lavage carried out 24 h after challenge with just saline or with allergen gave the same recovery in healthy control subjects and in asthmatics (Table 2). In the baseline fluid there was no difference in the total number of cells between the two groups (p = 0.69). After challenge with saline or with allergen, cells increased in the asthmatic group (p = 0.03) but not in healthy volunteers (p = 0.07). Allergen challenge in asthmatics caused a very significant increase in the number of eosinophils (p = 0.002) (Table 2).

Phospholipids and Proteins in BALF

As shown in Figure 1A the phospholipid concentrations in the cell-free supernatants from healthy subjects and from asthmatics were not significantly different, not at baseline or after the two types of challenge. The healthy subjects had low protein concentrations (Figure 1B) at baseline and there was no significant increase after challenge with saline or antigen. Also the asthmatics had a relatively low protein concentration at baseline and after saline challenge, but after antigen challenge there was a significant increase (p = 0.004, Figure 1B).

View larger version (48K): [in this window] [in a new window]   Figure 1.   Phospholipids and proteins in BALF. Phospholipid (panel A) and protein (panel B) concentrations in bronchoalveolar lavage fluids from healthy volunteers (N-) and from patients with mild atopic asthma (A-). -B indicates the BALF obtained at baseline, -S after challenge with saline, and A- after challenge with allergen. Values are given as mean ± SEM. ** indicate p < 0.01 compared with baseline.

When the cell-free supernatant of BALF was centrifuged at high speed, the supernatant contained the surfactant aggregates that were small and relatively surface inactive (SA) and the pellet the large surface active aggregates (LA). Phospholipid assays made it possible to obtain the ratio between SA and LA. As seen in Figure 2, that ratio remained low under all conditions, except after allergen challenge in asthmatics (p = 0.001).

View larger version (17K): [in this window] [in a new window]   Figure 2.   The ratios of surfactant aggregates. Ratio of small to large surfactant aggregates in BALF of healthy volunteers and asthmatics. The abscissa symbols are the same as in Figure 1. Values are given as mean ± SEM. ** indicate p < 0.01 compared with baseline.

Surface Activity Evaluated with the PBS

At baseline and after challenge with saline solution, surface tension at minimal bubble size after 5 min of pulsation was less than 2 mN/m in the healthy volunteers and in the asthmatics (Figure 3A). Challenge with antigen did not affect surface tension of the control subjects, but in the asthmatics it caused a significant increase over baseline values (p = 0.006). Also the adsorption rate (Figure 3B) was affected by allergen challenge in the asthmatics. Surface tension at the air-liquid interface of a bubble that had not been pulsating was 29.8 ± 2.9 mN/m 10 s after the bubble's creation, a significantly higher value than at baseline (p = 0.03).

View larger version (47K): [in this window] [in a new window]   Figure 3.   Surface activity evaluated with the PBS. Surface tension at minimal bubble size after 5 min of pulsation (min) increased significantly when the asthmatics had been challenged with allergen (-A) (panel A). Also, adsorption rate (ads), as judged from the value of surface tension at the surface of a nonpulsating bubble 10 s after its formation, was significantly affected in the asthmatics challenged with allergen (-B) (panel B). Values are given as mean ± SEM. * and ** indicate p values < 0.05 and 0.01, respectively, compared with baseline. Symbols along abscissa are the same as in Figure 1.

Surface Activity Evaluated with the CS

How the lavage fluids from the five healthy volunteers and from the 14 patients with mild asthma were able to maintain capillary patency after challenge is shown in Figure 4A. The fluid was concentrated 20 times, which causes the phospholipid concentration to be about 1 mg/ml. It can be seen that in the group of control subjects as well as in the asthmatics, challenge with saline solution brought down the value of "Open in %," but not significantly. However, when the asthmatics had been challenged with antigen, the lowering of the value was highly significant (p = 0.005 versus baseline).

View larger version (56K): [in this window] [in a new window]   Figure 4.   Surface activity evaluated with the CS. Results obtained when studying BALF with the CS. The value of "Open in %" when the BALF had been concentrated 20 times by centrifugation and dehydration (panel A). The depressed function noted in the panel A graph was probably mainly due to inhibitors since when the supernatant from -A was used to dilute CLSE to 1 mg/ml, the adverse effect of the supernatant was particularly noticeable when the BALF was from allergen-challenged asthmatics (panel B). When the inhibitors had been removed with a "washing" procedure the surfactant function of maintaining patency was restored in all samples but three of the allergen-challenged asthmatics (panel C ). The phospholipid concentration of those three samples was particularly low. Values are given as mean ± SEM. ** indicate a p value < 0.01. The symbols along the abscissa are the same as in Figure 1.

When the supernatant used to dilute CLSE to 1 mg/ml came from a sample of baseline BALF, there appeared to be no inhibitors since the CLSE function of maintaining patency was undisturbed. However, when the BALF was from antigen-challenged airways of asthmatics the presence of inhibitors became obvious (p < 0.01) (Figure 4B).

After the "washing" procedure, the surfactant function in most cases became normal since the value of "Open in %" became close to 100, indicating patency. However, three of the samples of BALF from antigen-challenged asthmatics remained at extremely low values, bringing down the mean value (Figure 4C). It turned out that the phospholipid values for those three samples were extremely low, less than 0.15 mg/ml, which explained the poor surfactant ability to maintain patency. However, the supernatants from those samples also had an abundance of inhibitors. When they were used to dilute CLSE to 1 mg/ml the value of "Open in %" was close to zero.

Correlation between Eosinophil Levels and Surfactant Function

When the asthmatics had been challenged with antigen the BALF had a higher content of eosinophils and a higher value of surface tension at minimal bubble size after 5 min of pulsation (_min). When the two values were plotted against each other (Figure 5A) a significant positive correlation was noted (r = 0.66, p = 0.01). A positive correlation was also noted when _min was plotted against protein concentration (r = 0.68, p = 0.007), protein/phospholipid ratio (r = 0.65, p = 0.01), and SA/LA ratio (r = 0.83, p = 0.0003).

View larger version (15K): [in this window] [in a new window]   Figure 5.   Correlation of eosinophilia with surface activity. Correlation of percent eosinophils with surface tension at minimal bubble size (min) (panel A) and with the value of "Open in %" that was obtained when the supernatant of the high-speed centrifuged BALF was used to dilute CLSE to 1 mg/ml (panel B).

In addition, when values of "Eos%" were plotted against the values of "Open in %" that were obtained when the supernatant of the high speed centrifugation was used to dilute CLSE to 1 mg/ml, a significant negative correlation was noted (r = 0.71, p = 0.005, Figure 5B). When "Eos%" was plotted against "Open in %" of concentrated BALF or against "Open in %" of concentrated BALF that had been "washed," the negative correlation was also significant (r = 0.54, p = 0.047 and r = 0.54, p = 0.045, respectively).

	DISCUSSION

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We hypothesized in the introduction that antigen challenge in a patient with asthma would lead to an inflammatory reaction with leakage of plasma proteins into the air spaces and, furthermore, a surfactant dysfunction. That assumption we found to be correct since when a single lung lobe of a patient with mild atopic asthma was challenged with a specific antigen, inflammatory cells, particularly eosinophils, invaded the airways as did plasma proteins, and when the BALF surfactant was tested, its function was shown to be hampered. Most likely, the reason for this was that proteins had invaded the airway and reached a tenfold increase in concentration. Proteins have extensively been proven to inhibit surfactant function (5), and the washing procedure that in most cases restored it removed water-soluble inhibitors such as the proteins. In addition, serum proteins have been shown to accelerate the conversion of well-functioning large surfactant aggregates to poorly functioning small surfactant aggregates (29), which might explain why we observed an increased SA/LA ratio. Moreover, large surfactant aggregate conversion is inhibited by surfactant protein A (30), and, interestingly, in patients with asthma the BALF levels of SP-A are depressed (14).

However, besides proteins, other inflammatory products might have caused the surfactant dysfunction also. Because there was a strong positive correlation between the number of eosinophils and surface tension at minimal bubble size (Figure 4) eosinophils or eosinophil cationic protein (ECP) might have inhibited the surfactant. A study by Young and colleagues (31) suggests that ECP is capable of forming stable transmembrane pores in phospholipid bilayers, a phenomenon that points to the possibility that ECP might interact also with surfactant layers and thus interfere with its function. Although surfactant is mainly arranged as a monolayer, it has been shown that the alveoli are probably, at least in part, coated with a multilayer of surfactant (32), which might serve as the potential target for ECP. Future studies are needed to investigate if and how eosinophils and their cell products such as granule proteins and lipid mediators interact with pulmonary surfactant.

With techniques available today it is impossible to selectively sample surfactant material that has originated from the short section of the terminal airways where blocking liquid columns might form. Of course, there is an uncertainty of the origin of the analyzed material. The surfactant obtained with the lavage procedure, as carried out in our study, was probably mainly of alveolar origin. Therefore, it seemed reasonable to evaluate the surfactant function with an instrument that simulates alveolar architecture and functionthe PBS. The results obtained clearly showed that when a single lung segment of a patient with mild atopic asthma was challenged with antigen, a disturbed surfactant function ensued. The disturbance indicated an increased risk of alveolar collapse at end- expiration since at minimal bubble size surface tension had not diminished as much as it normally does. Dysfunctioning surfactant that originates from the alveoli will not have changed when it reaches the conducting airways.

Most likely, it is in the narrowest sections of conducting airways that a poorly functioning surfactant would exert its most harmful effect. At that location it could increase the risk that liquid would accumulate and block airflow and, consequently, increase the mean resistance to airflow. The surfactant function that is likely to ensue in conducting airways was evaluated with the CS, which simulates the architecture and function of the terminal conducting airway. It might be argued that the surfactant evaluated for its function in conducting airways should have been harvested in those airways. It should be pointed out though that surfactant phospholipids, which clearly have been shown to exist in terminal conducting airways (33, 34), must have originated from the alveoli (35). When a high surface pressure has developed in the surfactant film lining the alveolus, part of the film is likely to be extruded into the respiratory bronchiole at end-expiration when surface pressure comes to a maximum.

The CS demonstrated, as did the PBS, that in most cases the BALF surfactant from antigen-challenged airways of patients with asthma was clearly dysfunctioning. In those cases the surfactant would probably not be able to fulfill its function of maintaining airway patency. That function was restored, however, in almost all instances when water-soluble inhibitors had been removed with "washing." Most likely the inhibitors consisted of proteins that had leaked into the airway, since their concentration was 10 times higher than normal. However, in three asthmatic patients who had been challenged with antigen, the BALF showed a severe surfactant dysfunction when tested with the CS ("Open in %" = 0.7, 3.8, and 5.0, respectively). Protein concentrations were exceedingly high in two of the three samples, which were 0.15, 6.78, and 4.91 mg/ ml, and when the supernatant, obtained from the centrifugation carried out to pellet the LA surfactant, was used to dilute CLSE the surfactant function was clearly inhibited ("Open in %" = 48.7, 7.4, and 6.9). However, protein inhibition was not the only reason for the extremely poor surfactant function since the "washing" procedure did not improve the function ("Open in %" = 2.5, 2.9, and 0.1, respectively). In those three cases the phospholipid concentration was so extremely low (< 0.15 mg/ml) that even if there had been no inhibitors the function would still be very poor. The low phospholipid concentration might be explained in part by a phospholipase hydrolysis or for other reasons a lack of pelletable phospholipids. This is in accordance with our data as we found the SA/ LA-ratios to be very high in those samples (1.3, 8.7, and 16.1).

Taken together, we have shown that local allergen challenge in patients with asthma leads to an inflammation of the air spaces with increased amounts of eosinophils and proteins, and a dysfunction of surfactant isolated from BALF. We suggest that a disturbance of surfactant function may be involved in the pathophysiology of the allergic asthma attack. Although our data do not directly prove the link between airflow obstruction and surfactant dysfunction, it seems reasonable to assume that during an asthma attack narrow conducting airways may become blocked, which might contribute to an increased airway resistance.