INTRODUCTION

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Lung disease in cystic fibrosis (CF) is characterized by repeated cycles of endobronchial infection and inflammation. These episodes result in the gradual destruction and fibrosis of small airways and the production of thick tenacious secretions, both of which contribute to airways obstruction and respiratory impairment.

Although the basic defect in CF has been established, it is still not clear how this leads to lung disease. It has been suggested that changes in the electrolyte composition of the airway surface liquid predispose to airway infection, possibly by impairing the activity of proteins involved in host defence at the airway surface (1).

Pulmonary surfactant is known to be present not just in the alveoli but also in the bronchioles (2), where it may have a number of functions, including maintaining the patency of small airways (3) and improving mucociliary clearance, through effects on both the viscosity of mucus (4) and the ciliary beat frequency (2, 5). Other possible functions of surfactant include a role in host defence. Surfactant protein A (SP-A) has been shown to be an activator of macrophages and to enhance macrophage phagocytosis of a number of bacteria, including Staphylococcus aureus and Pseudomonas aeruginosa (6). It has also been suggested that surface active phospholipids, as well as being present at the air-surface interface, may bind to airway epithelial cells and form a protective layer (7). Several studies in adults with CF have documented either an abnormal quantity of total phospholipid (PL) or abnormal phospholipid composition of bronchial secretions (8, 9).

Some of these changes have been associated with increases in mucus rigidity, or increased mucus adherence to the respiratory mucosa. The surface activity of surfactant obtained from adult CF subjects by bronchoalveolar lavage (BAL) has been shown to be reduced (10).

These reported alterations in surfactant composition and activity could each contribute to the lung disease found in patients with CF, and trails of aerosolized surfactant preparations as therapy for CF lung disease are under way (11). However, it is not clear whether the observed alterations in surfactant are a primary abnormality in these subjects caused, for example, by alterations in the electrolyte content of the airway surface liquid or whether they are consequences of airway infection and inflammation.

The purpose of the present study was to determine whether surfactant composition and function are initially normal in infants with CF and to test the hypothesis that alterations in surfactant only occur in the presence of infection and/or inflammation.

	METHODS

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Subjects and Controls

The state of Victoria, Australia (66,000 births/yr) has a newborn CF screening program. The diagnosis is confirmed by sweat chloride concentrations of greater than 60 mmol/L and all patients are managed by the Royal Children's Hospital CF Clinic. BAL samples were collected from asymptomatic infants with CF identified by screening and from infants and young children with CF during episodes of respiratory exacerbation. A total of 27 infants and young children with CF, with a mean age 22.7 mo (SD 14.5, range 2-54), were investigated. Six children undergoing bronchoscopy for stridor formed a non-CF control group with a mean age of 23.8 mo (SD 21.7, range 2-54). The controls did not have signs of respiratory infection or a history of antibiotic use during the previous 14 d.

BAL fluid from all subjects was examined for the presence of infection by quantitative culture and for inflammation by cytology and interleukin-8 (IL-8) analysis as described previously (12). Cultures  10⁵ colony forming units, IL-8 levels above 250 pg/ml BAL fluid and the proportion of neutrophils present above 50% were taken to indicate the presence of infection and inflammation. On the basis of these markers, the CF subjects were divided into two groups, one group in which each subject had at least two out of the three markers of pulmonary infection or inflammation present (n = 12, CF-I) and a second group in which all three markers were negative (n = 15, CF-NI). Clinical details of the two CF groups and the controls are given in Table 1.

The details of the BAL fluid cytology, culture and IL-8 measurements for the CF and control patients are shown in Table 2. In the CF-I group, BAL fluid from nine of 12 subjects had a significant growth on quantitative culture (three P. aeruginsa, three S. aureus, two Moraxella catarrhralis, and one Haemophilus influenzae).

Bronchoalveolar Lavage

BAL was performed under general anaesthesia. Following topical application of lignocaine to the vocal cords, a flexible bronchoscope (Olympus model BF3 C20; Olympus Optical Co., Ltd., Japan; external diameter, 3.6 mm; suction channel, 1.2 mm) was introduced into the lower airway through a laryngeal mask, avoiding the use of the suction channel until the tip of the bronchoscope was below the carina. The tip was wedged in the right middle lobe bronchus and to optimize sampling from endobronchial sites, a single small volume lavage was performed by instilling 1.5 ml/kg of nonbacteriostatic normal saline through the bronchoscope over three to five seconds. The saline was immediately aspirated into a suction set over 10-20 s using negative suction pressures of 100-150 mm Hg. The bronchoscope was then wedged into the lingula bronchus and using identical technique a further single aliquot lavage was performed. The BAL fluid from both lavages was pooled for analysis, placed on ice and taken to the laboratory for processing. None of the BAL samples collected had macroscopic evidence of blood contamination.

Surfactant Studies

BAL to be used for surfactant studies was centrifuged at 150 g for 5 min at 4° C to removed cells and debris. The supernatant was then stored at 70° C until analysis. All analyses were performed with the investigator blinded to the origin of the sample.

Analysis of lipids. Lipids were extracted from the BAL fluid by the method of Bligh and Dyer (13). The disaturated phospholipids (DSP) were separated by the method of Mason and colleagues (14) and the PL content was determined by measuring inorganic phosphorus by the Bartlett method (15). All measurements were performed in duplicate.

SP-A and SP-B analysis. SP-A and surfactant protein B (SP-B) were measured using the inhibition enzyme linked immunosorbent assays developed by Doyle and Nicholas (16, 17). To free the SP-A and SP-B from an associated surfactant components, 125 µl aliquots of lavage supernatant were diluted in 500 µl of 10 mM tris, 1 mM ethylenediaminotetra acetic acid (EDTA) containing 0.25% bovine serum albumin (BSA) pH 7.4. After vortexing at room temperature for 10 min, 125 µl of solution containing 3% SDS and 12% Triton X-100 (vol/vol) was added to each and the samples again vortexted for 10 min. The SP-A and SP-B ELISAs were then performed as described elsewhere (16, 17).

Surface tension measurements. For measurement of surface functional activity, BAL fluid was centrifuged at 25,000 g at 4° C for 1 h, producing a lipid pellet which was resuspended in buffer (140 mM NaCl, 10 mM HEPES, 0.5 mM EDTA, 2.5 mM CaCl₂ at pH 6.9) and adjusted to DSP concentration of 1 mM (750 microgram/ml DSP). The surface activity of the resuspended pellet was then assessed using the pulsating bubble surfactometer (Electronetics Corporation, Amherst, NY) (18). Measurements of minimum surface tension (ST) were made during adsorption and compression as described previously (19).

Estimation of Epithelial Lining Fluid

As a marker of dilution of the epithelial lining fluid (ELF) urea was measured in the serum (taken at the time of the BAL) and BAL fluid of each patient. Serum urea was measured using the Ektachem 250 Analyzer (Johnson and Johnson Clinical Diagnostics Inc., Rochester, NY). BAL urea was measured using a standard urease/glutamate dehydrogenase kit (Sigma 66-UV; Sigma Chemical Company, St. Louis, MO) adapted to analyze urea in micromolar concentrations using a Cobas Bio centrifugal analyzer.

Statistics

Since data were not normally distributed, nonparametric methods were used in data analysis, including the Mann-Whitney U statistic for the between group comparisons. The results for the lipid and surfactant protein analysis were expressed in micrograms per milliliter of BAL fluid and, after correction using urea levels from BAL fluid and serum, as micrograms per milliliter of ELF. In addition, the surfactant proteins were expressed as ratios with the major surface active lipid component of surfactant, DSP.

	RESULTS

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The phospholipid composition and SP-A and SP-B concentrations of bronchial secretions from the CF subjects without evidence of lower respiratory tract infection or inflammation (CF-NI group) did not differ significantly from that found in the control subjects (Table 3). When the minimum (ST) was measured at a fixed concentration of DSP (1 mM), there was no significant difference between the CF-NI subjects and controls, either after 10 min adsorption or 10 min compression at 20 cycles/s (Table 4).

Phospholipid analysis of the bronchial secretions from the CF group with pulmonary infection or inflammation (CF-I) showed that although there were no significant differences in the concentrations of total PL or DSP (per ml of BAL fluid or corrected for ELF), the percentage of PL as DSP was significantly reduced, both compared with BAL fluid from the CF-NI group (p = 0.05) and to that from controls (p = 0.003) (Table 3). Minimum ST measurements at 1 mM DSP for the bronchial secretions from the CF-I subjects did not differ from those found in the controls or the CF-NI subjects (Table 4).

The SP-A concentrations in BAL fluid, and when expressed as a ratio to DSP, were significantly higher in the CF-I group when compared to both the control group and the CF-NI group (p < 0.05 for both parameters). When the SP-A concentration was corrected to ELF volume using urea, a trend toward a higher value in the CF-I group was still evident but it failed to reach statistical significance (Table 3). Analysis of SP-B showed a trend in the opposite direction with levels of SP-B tending to be lower in the CF-I group. However these differences did not reach statistical significance, either for SP-B expressed per milliliter of BAL fluid, per milliliter ELF, or as a ratio to DSP (Table 3).

As would be expected from the natural history of the disease, the CF-I group were significantly older than the CF-NI group (mean age ± SD: CF-I 31.7 ± 15.4 mo; CF-NI 15.5 ± 8.8 mo) with the control group falling between these two (23.8 ± 21.7 mo). There was no statistically significant effect of age (by linear regression) on any of the parameters within the control group.

	DISCUSSION

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In this study, we have shown that, in the absence of lower respiratory tract infection or inflammation, there were no significant differences in the composition or surface activity of bronchial surfactant from infants and young children with CF compared with those from a similarly noninflammed nor infected non-CF control group. In contrast, in infants and young children with CF who had evidence of infection and/or inflammation in BAL fluid, there was a fall in the DSP to PL ratio and an increase in the concentration of SP-A. These results suggest that the abnormalities in surfactant function and composition found in CF subjects in this study and those found in previous studies are a secondary, rather than a primary, abnormality of CF.

The changes in surfactant lipid composition found in the present study are similar to those reported for adult CF subjects. Gilljam and colleagues collected bronchial samples from seven adult subjects with CF (8). The most significant abnormality they found was a reduction of the phosphatidylcholine (PC) fraction of total PL, particularly in those patients with more severe disease. Our finding that the DSP to PL ratio was reduced in the CF group with pulmonary inflammation or infection (CF-I) supports this result (since almost all the DSP is in the PC fraction). The mechanism by which the DSP to PL ratio is lowered is not known. It is possible that phospholipases secreted by bacteria are responsible. Holm and colleagues have shown that pulmonary surfactant is inhibited by phospholipases (20). One of these phospholipases, PLA₂ has its primary action dipalmitoyl-phosphatidylcholine (DPPC). We found that the minimum ST achieved at 1 mM DSP did not differ significantly among the three study groups. Measuring ST at a constant DSP concentration means that any variation of ST does not simply reflect differing concentrations of DSP but rather the presence of inhibitors of surfactant function. Gilljam and colleagues (9) also measured ST in their samples and found very similar results to those reported here (minimum ST of 24.6 mN/m in CF subjects and 23.2 mN/m in controls). The reason that the majority of the samples from the control group in the present study and from the one of Gilljam and colleagues gave relatively high minimum ST readings (alveolar surfactant from normal lungs gives a minimum ST on compression of less than 5 mN/m), is likely to be because the samples were bronchial rather than alveolar, and presumably contain either some inhibitors of surfactant function or lack some surface active components. Meyer and colleagues (10) were able to show a difference between the CF patients with P. aeruginosa infection and normal controls. The minimum ST in their CF patients was 21.9 mN/m and less than 1 mM/m in controls. Details of the method of sample collection were not given, but it is likely that a multiple aliquot technique was used. In normal subjects this will sample the alveolus, but in adults with CF and small airway obstruction, it is likely that the sample is predominantly bronchial. Furthermore these samples were measured at a fixed concentration of total PL rather than DSP. Since the DSP to PL ratio is reduced in CF then the CF samples would also have contained less DSP than the controls.

In the present study, SP-A, whether expressed as micrograms per milliliter of BAL fluid or expressed as ratios with DSP, was significantly higher in the CF-I group than in both the CF group with no pulmonary inflammation (CF-NI) and the control group. When the SP-A concentration was corrected for ELF volume using urea, the CF-I group still had the highest levels although this failed to reach statistical significance. In contrast, SP-B was lower in the CF-I group compared to the other groups but did not reach statistical significance. The subjects in the CF-I group were on average several months older than those in the other two groups. Within the control group, there was no effect of age on the DSP:PL ratio or on the SP-A and SP-B levels. Furthermore, the available data on children suggests that SP-A concentrations fall with age (21). Thus the finding of an increased level of SP-A in the older CF-I group is likely to be more rather than less significant if age is taken into account.

In more pulmonary diseases studied, including bacterial and fungal pneumonia, (22, 23) SP-A levels have been lower than in controls. One study has shown an increase in SP-A levels in HIV positive adults with Pneumocystis carinii pneumonia compared to HIV-positive patients without infection, although in both groups of patients the SP-A levels were lower than in control subjects (24). The present study is the first to show higher SP-A levels in response to pulmonary infection and associated inflammation compared to controls, particularly whilst other components of surfactant are either unchanged or reduced. This suggests a role of SP-A in this situation that is not related to the surface activity of surfactant. The most likely alternative function for SP-A would be one of host defence. SP-A is secreted by alveolar type 2 cells, bronchiolar Clara cells (25) and by the epithelium and glands of the large airways (26). We speculate that the low grade persistent nature of endobronchial infection in CF may provide a much stronger stimulus to SP-A secretion than acute pneumonia. Recent work using SP-A "knock-out" mice provides further support for the host defence function of SP-A and suggests that it may have little effect on the surface activity of surfactant in vivo (J. A. Whitsett, personal communication). These mice produce no SP-A and yet have normal lung compliance. However, they have an increased susceptibility to pulmonary bacterial infection.

In summary, we have found that in the absence of active lung disease, the surfactant indices of bronchial secretions of CF infants and young children do not differ significantly from those of children without CF. In contrast, in the presence of infection or inflammation there is a change in the lipid and protein composition of these secretions. The DSP to PL ratio falls, which is likely to reduce the surface activity of the bronchial surfactant, and the SP-A level is increased above the concentrations found in control subjects. We suggest that the increased levels of SP-A are in response to endobronchial infection and support a role for SP-A in host defence.