Published ahead of print on July 21, 2004, doi:10.1164/rccm.200405-575OC
© 2004 American Thoracic Society doi: 10.1164/rccm.200405-575OC
Pulmonary Surfactant, Lung Function, and Endobronchial Inflammation in Cystic FibrosisChildren's Hospital, University of Munich, Munich; Internal Medicine, University of Giessen, Giessen; Department of Pediatric Pneumology and Allergology, Children's Hospital, University of Cologne, Cologne; Children's Hospital, University of Essen, Essen; Department of Pediatric Pneumology, Medical School, University of Hannover, Hannover; and Department of Pediatric Pneumology and Immunology, Charité, Humboldt-University, Berlin, Germany Correspondence and requests for reprints should be addressed to Professor Matthias Griese, M.D., Children's Hospital, University of Munich, Lindwurmstr 4, 80337 Munich, Germany. E-mail: matthias.griese{at}med.uni-muenchen.de
Cystic fibrosis (CF) lung disease is primarily a disease of the small airways. We hypothesized that even in patients with normal lung function, a reduced surfactant function would be present and favor small airway obstruction. Bronchoalveolar lavages from 76 patients with CF (5–31 years, median 11) with well-conserved lung function (FEV1 94% predicted, range 78–121) and from 10 healthy control subjects were investigated. The deviation of the biophysical surfactant performance from normal, assessed in a bubble surfactometer, was small; however, the ability of the surfactant to maintain the patency of a narrow airway (% open) was significantly reduced. Surfactant protein (SP)-C level was increased, SP-B and SP-D were unchanged, whereas SP-A was decreased. Among the patients with CF, neutrophilic inflammation was modestly related to a poorer surfactant activity, but not to lung function. SP-D was reduced in proportion to the degree of inflammation and in the presence of bacteria. These findings in a large cohort of patients with CF with normal lung function show that the endobronchial airway inflammation is linked to early perturbations of the biophysical properties and immunologic components of pulmonary surfactant and opens fields for novel therapeutic interventions.
Key Words: airway inflammation • bronchoalveolar lavage • capillary surfactometer • pulsating bubble • surface activity Cystic fibrosis (CF) is primarily a disease of the small airways, and early alterations caused by mutations in the CF transmembrane regulator gene are found in this region of the respiratory tract (1). Multiple lung function tests have been used to assess these early functional abnormalities, but none of them has been found to be particularly sensitive (2, 3), and in patients with normal pulmonary function tests, pathologic changes like structural abnormalities (4, 5) and lower airway inflammation may be present (6, 7). Pulmonary surfactant plays a pivotal role for the biophysical and immunologic integrity of the lungs (8, 9). The surfactant proteins (SPs) B and C, together with the surfactant lipids, form a well-functioning system that maintains the patency of the small airways and alveoli (10, 11). An altered surfactant-phospholipid composition is associated with impaired lung function in young children with respiratory infections (12). Thus, derangements of any of these components may affect the biophysical surfactant activity and may contribute to early alterations of the lung function in patients with CF. In infants and young children, both in the presence and absence of airway infection, the surface activity of surfactant, recovered by bronchoalveolar lavage (BAL) and assessed in a pulsating bubble surfactometer, was found to be unaltered (13). However, in children with CF, median age 7.5 years (range 1–16), with somewhat impaired lung function (FEV1 82% of predicted [range 40–103]), minimal surface tension was reduced, whereas the concentrations of total phospholipids and of dipalmitoyl phosphatidylcholine were within the normal range (12, 14). The same was found in older children and young adults (15), suggesting that there may be an early, but secondary, derangement of the biophysical surfactant properties in CF. However, surfactant function with respect to its specific role for small airways patency are as yet unknown. As the early abnormalities in CF involve small airways, we hypothesized that the major abnormality in the surfactant system that contributes to CF lung disease is its reduced function, which would favor small airway collapse or obstruction by airspace liquids and could be best assessed in a capillary surfactometer. In addition to the biophysical aspects of surfactant, the two collectins, SP-A and SP-D, which bind endotoxin and glycoconjugates on the surface of various viral, bacterial, and fungal pathogens are important regulators of the inflammatory state of the lungs (8, 16, 17). Mice made genetically deficient for SP-A and SP-D demonstrate a poor clearance of microorganisms and have prolonged and increased lung inflammation after microbial exposure. Primary or secondary deficiency states of these collectins may therefore affect the clearance and persistence of lower airway inflammation. Previous studies have reported alterations in these collectins in patients with CF. SP-A was increased in infants and young children irrespective of the presence or absence of pulmonary inflammation (13), whereas in older children and adults with CF, SP-A was consistently decreased (14, 15, 18). In addition, proteolytic degradation of SP-A in CF has been shown, supporting the concept that inflammation may play a role in decreasing SP-A function in CF airways (19, 20). SP-D has been shown to be reduced in young patients with CF (21) as well as in patients with CF with more advanced lung disease (14). However, both groups of patients were assessed during periods of clinically active lung disease, and it is presently unknown whether these decreased concentrations persist in patients who are clinically stable. The goal of the present study was to characterize the biophysical and biochemical properties of pulmonary surfactant in a large cohort of patients with CF with relatively well-conserved lung function and a stable clinical condition. Our hypothesis was that surfactant function, assessed in a capillary surfactometer and representing the small airways where early inflammatory processes may perturb the biophysical surfactant properties, will be impaired long before alterations of lung function may be noticed. Additionally, we were interested in the concentrations of the surfactant collectins under these conditions. Some results of these studies have been reported previously in the form of an abstract (22).
Patients and Samples All samples from patients with CF were recruited from the bronchoalveolar lavage for the evaluation of antiinflammatory treatment (BEAT) study, a large multicenter study using BALs to evaluate the effect of rhDNase (Pulmozyme; Roche, Grenzach, Germany) on endobronchial inflammation (23). Previous inclusion criteria were the diagnosis of CF, the ability to perform lung function tests, a normal lung function defined as a FEV1 greater than 80% predicted, and a clinically stable condition. Exclusion criteria were the application of antiinflammatory treatment, like ibuprofen and systemic or inhaled corticosteroids, and bronchopulmonary aspergillosis. All patients had to be free of acute respiratory tract infections for at least 6 weeks before bronchoscopy. The study was approved by the local ethic committees of all participating centers. Written informed consent by both parents and/or the patients was obtained in all cases. For the present study, samples from all patients not having received rhDNase were considered. Figure 1 lists details regarding excluded and included patients. Seventy-six patients with CF were included (median age 11 years, range 5–31; females/males, 41/35. Forty-seven of the patients were dF508 homozygeous, 16 were dF508 compound heterozygeous, in five patients other mutations were present, and in eight the mutations were unknown. Other characteristics: 19 of the 76 patients were Pseudomonas aeruginosa positive; FVC, 97% predicted, range 70–126; FEV1, 94% predicted, range 78–121; and MEF25%VC, 61% predicted, range 18–155. The BAL fluid (BALF) from these patients was compared with that from 10 healthy children (median age 4.5 years, range 1.5–8.5) previously collected (24) and stored in aliquots at –80°C for analysis.
BAL, Sample Preparation, and Biochemical and Biophysical Measurements BAL was performed as described before with 3 ml/kg body weight normal saline warmed to body temperature (23, 25). The first aliquot of the recovered BALF was treated separately; all other samples were pooled for analysis. The total cell count was measured in a hemocytometer, and the differential cell count was assessed from cytoprep slides. Aliquots of the cell-free BALF supernatant were used directly for the analysis of total protein and the surfactant proteins (SP-A, SP-D [26] and SP-B, SP-C [15, 27]) by ELISA. Five-milliliter aliquots of the BALF were centrifuged at 40,000 x g for 30 minutes to generate a surfactant pellet containing the large surfactant aggregates (15). The phospholipid content was determined by phosphorus assay of a lipid extract of the surfactant pellet (15). After resuspension of the surfactant pellet (140 mM NaCl, 10 mM N-2-hydroxyethylpiperazine-N'-ethane sulfonic acid [HEPES], 0.5 mM ethylenediamimetetraacetic acid [EDTA], 3.5 mM CaCl2, pH 6.9), its surface activity was assessed in a pulsating bubble surfactometer (Electronetics, Buffalo, NY) at a final phospholipid concentration of 1 mg/ml (15). In addition, the biophysical activity of the surfactant material was assessed in a capillary surfactometer (Calmia Medical, Toronto, ON, Canada) (28). Briefly, glass capillaries (0.255 mm internal diameter at its most narrow part) were filled with 0.5 µl of the above described surfactant suspension and assessed for its capability at 37°C to keep the capillary open over a time period of 120 seconds. H2O (0% of the time open) and a purified bovine surfactant (96.7% of the time open) served as controls (28).
Statistical Analysis
Comparison of Patients with CF with Healthy Control Subjects BALF concentrations of phospholipid and surfactant protein concentrations are shown in Table 1. As reported previously, patients with CF had an increased percentage of neutrophils and increased absolute numbers of neutrophils and total cells recovered by lavage (Table 1). This neutrophilic inflammatory reaction was associated with decreased concentrations of phospholipids and by an increased concentration of total protein. Among the surfactant protein concentrations, SP-A was significantly decreased, SP-C was marginally increased, and the other two surfactant proteins were not changed (Table 1). The biophysical activity of the surfactant recovered from the lungs of the patients with CF as assessed in the pulsating bubble surfactometer showed a tendency to lower surfactant function, which failed to reach statistical significance (Figure 2, left panel). When assessed in the capillary surfactometer, significant differences were obvious (Figure 2, right panel).
Lung Function, Surfactant Function, and Components and Degree of Inflammation Differ with the Presence or Absence of Microorganisms within the Cohort of the Patients with CF In 57 of the patients with CF, bacterial pathogens were recovered in their BALF. These included the following characteristic CF-related pathogens: Staphylococcus aureus (n = 30), Haemophilus influenzae (n = 17), P. aeruginosa (n = 19), other bacteria (n = 15), Aspergillus species (n = 3), Candida species (n = 7), and other fungi (n = 2). This group of patients did not differ in age, sex, and body mass index from the patients without pathogens recovered in their BALF. However, the patients without pathogens had better midexpiratory flows, a superior biophysical activity of their surfactant, and less inflammation (Table 2). Whereas SP-A was not different, SP-D was decreased (Table 2). In a comprehensive further analysis, patient subgroups with the different bacteria recovered were compared. No differences for any of the variables investigated were obtained when results for patients with S. aureus or P. aeruginosa or both were compared with each other or with patients with the other microorganisms recovered (data not shown). Lung function, assessed as FEV1, FVC, and MEF25–75%VC, did not correlate with any of the biophysical variables, the inflammatory variables, the surfactant proteins, total protein, or the phospholipids recovered (data not shown).
Surfactant Function and Surfactant Components Do Not Predict Lung Function, but Correlate with Inflammation The biophysical performance of the surfactant from the patients with CF consistently correlated with the degree of airway inflammation, as assessed by BAL percentage of neutrophils (Figure 3). Similar results were obtained for the absolute numbers of BAL cells and the absolute neutrophil counts in BALF (data not shown). Decreased minimal surface tension, i.e., a worse surfactant function, was associated with less SP-A (r = –0.273, p = 0.021). The concentration of SP-A was linked to that of SP-B and the phospholipids (r = 0.300, p = 0.017 and r = 0.348, p = 0.002). Very similar to SP-A, a worse surfactant function was associated with less SP-D (Figure 4), and a larger percent openness of the tube was related to higher levels of SP-D (r = 0.335, p = 0.003). SP-D concentration decreased with increasing absolute amounts of neutrophils (r = –0.330, p = 0.005) or total lavage cells (r = –0.330, p = 0.003) and the percentage of neutrophils (Figure 5).
This study of a large and well-defined population of patients with CF assesses for the first time the relation of endobronchial inflammation, biophysical surfactant activity, the level of all surfactant proteins, and lung function in nonventilated, clinically stable subjects. Most strikingly, increased neutrophilic inflammation was modestly related to poorer biophysical surfactant activity, but not to FEV1 and FVC. These findings add to the hypothesis that the endobronchial airway inflammation may directly perturb the pulmonary surfactant system in CF and this may contribute to early obstructive lung disease. In this cohort of patients with CF with lung function within the normal range, the impairment of the biophysical surfactant performance, assessed as minimal surface tension and surface tension after adsorption in the pulsating bubble surfactometer, and representing a model for the alveolar setting, was somewhat worse, but not significantly different from that of the controls. In contrast, the ability to maintain the patency of a narrow airway, as assessed in the capillary surfactometer, was greatly impaired in a substantial number of subjects. The results provide evidence for the model of an inflammation-dependent loss of surfactant function in the smaller airways. Such a loss of stability of the peripheral airways, with little or no reduction of surface activity in alveoli, will lead to increased airway resistance, earlier airway closure at high lung volumes, and gas trapping (29). Less appreciated, but possibly even more important, is the prevention of epithelial cell damage during airway reopening. A well-functioning surfactant greatly minimizes reopening pressures and, by this, airway wall stresses and cellular injury (30). The biochemical mechanisms responsible for the impaired surfactant function include increased total protein content in the CF samples. Increased protein has previously been shown in the BALF from patients with CF with more severe lung disease (15), and is also a major causal factor for impaired surfactant function in this and other pulmonary diseases (9, 11). However, in this study no correlations between lung function variables or surfactant function were found, making a major contribution of this mechanism unlikely. Decreased concentrations of SP-A, but not SP-D may also contribute to impaired biophysical activity (18, 31). A deficiency of SP-B or SP-C in these patients with CF with mild lung disease can be excluded on the basis of our data. In accordance with the elevated level of SP-C obtained here, we previously found increased SP-C levels in children with malignancies, fever, and chest infiltrates, who also had a neutrophilic inflammatory response in their BAL fluid (32). SP-C may well contribute to the stabilization of the surface activity of the surfactant (33). An altered phospholipid composition may also contribute to reduced surface activity, but that appears rather unlikely, as no significant deviations have previously been found in a group of younger patients with CF with somewhat worse, but still relatively well-preserved lung function (12, 14). Lastly, a systematic failure to recover well-functioning surfactant components by BALF from mucous-plugged and inflamed airways might also result in a loss of surfactant activity and an apparent correlation between inflammation and decreased surfactant function. Although unlikely to be the sole reason, such an explanation may be addressed in future studies. The physiologic consequences of the perturbation of the airway surfactant system at this disease state were not detectable by conventional lung function testing, i.e., FEV1, FVC, or MEF25. However, surfactant activity was consistently correlated to the degree of neutrophilic inflammation and a reduction in biophysical activity. These findings are similar to the discrepancy of often normal or only mildly reduced pulmonary function testing in young children with CF, where there is at the same time an exuberant inflammatory response and increased level of deoxyribonucleic acid in the airspaces (7, 34). The lung collectins, SP-A and SP-D, have dual regulatory functions within the pulmonary host defense system, i.e., suppression or enhancement of inflammation (17). Previously, we and others have demonstrated that SP-A and SP-D were reduced in several studies of patients with CF (13–15, 21). In agreement with these data, in this large sample set of much older patients with preserved lung function, we also found reduced levels of SP-A. Of interest, overall, the SP-D concentration was not significantly decreased. This finding is likely explained by less infection and inflammation in these patients compared with the children investigated by Noah and colleagues (21) during a phase of more active disease. In accordance, we found within the group of patients with CF, that those with bacteria recovered in their BAL also had reduced SP-D levels (Table 2), similar to that previously observed in children with tracheostoma (24). As we did not investigate SP-D levels in lavage cells or the expression of SP-D in lung tissue, we cannot comment on the mechanisms involved. Proteolytic degradation by neutrophil-derived proteases may occur in lungs of patients with CF (35, 36), however, this was unlikely to be the cause here, as we did not observe the characteristic degradation products in a small subset of lavages, even in those with a high level of neutrophils (data not shown). Irrespective of the cause, a deficiency of SP-A and SP-D may contribute to the neutrophil-dominated inflammation by failure to clear the microorganisms by phagocytosis and subsequent killing (37, 38), which has also been shown for genetically engineered SP-A and SP-D deficient mice (39). On the other hand, reduced levels of the collectins in the presence of an overwhelming chronic burden of cellular debris and continuous bacterial exposure, as present in patients with CF, may be an appropriate adaptation. When the collagenous tails of the collectins after binding to debris or bacteria are presented, they can bind to calreticulin/CD91 receptors on macrophages or pulmonary epithelial cells and initiate a proinflammatory response. Thus, low collectin levels in the presence of large amounts of pathogens or cellular debris may protect the lung from perpetuating the proinflammatory cascade (17). The role of the lung collectins in chronic states of airspace inflammation needs further evaluation to clarify if their supplementation may be a reasonable therapeutic intervention. In summary, in a large cohort of patients with CF with normal lung function, we showed that endobronchial inflammation is linked to impairments of the biophysical surfactant properties to keep small airways patent, e.g., reduced percentage of open values, and its immunological components, e.g., reduced levels of SP-A and SP-D. Therapeutic interventions to counterbalance such alterations may open novel strategies for treatment.
The authors thank all the staff in the clinics and laboratories at the different participating centers for their excellent collaboration in this study.
Supported by a grant from the German Cystic Fibrosis Foundation (Mukoviszidose e. V.), Hoffmann-La Roche, Germany, and a grant from the Deutsche Forschungsgemeinschaft (Gr 970/7-1). M.G., E.R., F.R., M.B., and K.P. are principal investigators of the BEAT study group. The manuscript contains parts of the medical thesis of Robert Essl.
Conflict of Interest Statement: M.G. does not have a financial relationship with a commercial entity that has an interest in the subject of this article; R.E. does not have a financial relationship with a commercial entity that has an interest in the subject of this article; R.S. does not have a financial relationship with a commercial entity that has an interest in the subject of this article; E.R. does not have a financial relationship with a commercial entity that has an interest in the subject of this article; F.R. does not have a financial relationship with a commercial entity that has an interest in the subject of this article; M.B. received $1,200 in 2004 for speaking at conferences sponsored by Hoffman La-Roche; K.P. has received Received in original form May 1, 2004; accepted in final form July 16, 2004
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1,000 for lectures from Hoffmann La-Roche Company in 2001, 2002, 2003, and 2004. 
or calreticulin/CD91, lung collectins act as dual function surveillance molecules to suppress or enhance inflammation. Cell 2003;115:13–23.
