Conductive Airway Surfactant: Surface-tension Function, Biochemical
Composition, and Possible Alveolar Origin
Wolfgang Bernhard, Henk P. Haagsman, Thomas Tschernig, Christian F. Poets, Anthony D. Postle,
Martin E. van Eijk, and Horst von der Hardt
Departments of Pediatric Pulmonology and Anatomy, Medical School Hannover, Hannover, Germany; Department of
Veterinary Biochemistry, Utrecht University, Utrecht, the Netherlands; and Department of Child Health,
Southampton General Hospital, University of Southampton, Southampton, United Kingdom
Alveolar surfactant is well known for its ability to reduce minimal surface tension at the alveolar air–liquid
interface to values below 5 mN/m. In addition, it has been suggested that an analogous conductive airway
surfactant is also present in the airways. To elucidate the composition, possible origin, and surface activity
of conductive airway phospholipids (PL), we compared in adult porcine lungs the PL classes and
phosphatidylcholine (PC) molecular species of nonpurified tracheal aspirate samples with those of bronchoalveolar lavage fluid (BAL), tracheobronchial epithelium, and lung parenchyma. We also analyzed PL
and PC composition, protein content, and surface activity of surfactant isolated from tracheal aspirates
(SurfTrachAsp), BAL (SurfBAL), and the 27,000 3 g pellet of BAL (SurfP27000) by density-gradient centrifugation. Although PL composition revealed contributions of the airways to tracheal aspirates, the composition
of PC molecular species of tracheal aspirates was similar to that of BAL and lung parenchyma, but differed
considerably from that of airway epithelium. SurfTrachAsp had the same PL and PC composition as SurfBAL
and SurfP27000, indicating that this fraction of tracheal aspirates may have originated from the alveoli. Nevertheless, minimal and maximal surface tensions were higher in SurfTrachAsp than in SurfBAL and SurfP27000.
Analysis of surfactant proteins A, B, and C (SP-A, SP-B, and SP-C) revealed that SP-A was decreased and
SP-B and SP-C were absent, whereas total protein was increased in SurfTrachAsp. We conclude that as compared with alveolar surfactant, PL of SurfTrachAsp show the same composition, but that surface-tension
function is impaired and the concentration of surfactant proteins is decreased in SurfTrachAsp. Bernhard,
W., H. P. Haagsman, T. Tschernig, C. F. Poets, A. D. Postle, M. E. van Eijk, and H. von der Hardt.
1997. Conductive airway surfactant: surface-tension function, biochemical composition, and possible
alveolar origin. Am. J. Respir. Cell Mol. Biol. 17:41–50.
Pulmonary surfactant, a mixture of phospholipids (PL)
and proteins lining the alveolar epithelium, is essential for
normal alveolar function (1). Phosphatidylcholine (PC) of
whole lung is highly saturated and largely reflects the com(Received in original form March 26, 1996, and in revised form November
4, 1996)
Address correspondence to: Wolfgang Bernhard, Ph.D., Department of
Pediatric Pulmonology, Medical School Hannover, Konstanty-GutschowStrasse 8, 30625 Hannover, Germany.
Abbreviations: 1,6-diphenyl-1,3,5-hexatriene, DPH; high-performance liquid
chromatography, HPLC; lysophosphatidylcholine, LPC; phosphatidylcholine, PC; palmitoylmyristoyl PC, PC16:0/14:0; dipalmitoyl PC, PC16:0/16:0;
palmitoyl-palmitoleoyl-PC, PC16:0/16:1; palmitoyloleoyl PC, PC16:0/18:1;
palmitoyllinoleoyl PC, PC16:0/18:2; palmitoyllinolenolyl PC, PC16:0/18:3;
palmitoylarachidonoyl PC, PC16:0/20:4; palmitoyldocosahexaenoyl PC,
PC16:0/22:6; stearoyllinoleoyl PC, PC18:0/18:2; stearoylarachidonoyl PC,
PC18:0/20:4; dioleoyl PC, PC18:1/18:1; oleoyllinoleoyl PC, PC18:1/18:2;
phosphatidylethanolamine, PE; phosphatidylglycerol, PG; phosphatidylinositol, PI; phosphatidylserine, PS; sphingomyelin, SPH.
Am. J. Respir. Cell Mol. Biol. Vol. 17, pp. 41–50, 1997
position of alveolar surfactant and of type II alveolar epithelial cells that synthesize surfactant (1). The presence of
PL and protein in bronchial secretions has been well documented, and one of their major postulated functions in
such secretions is to inhibit adhesion of the ciliae to the
mucous gel and, by accelerating ciliary beat frequency, to
make the gel phase glide on top of the sol phase toward
the pharynx (2–6). However, the source of this material is
not known. Although it is clear that no cell type of the
conductive airway epithelium contains lamellar inclusion
bodies characteristic of surfactant stored in type II alveolar epithelial cells, Clara cells synthesize surfactant proteins A, B, and D (SP-A, SP-B, and SP-D) (7–9). No previous analysis has characterized conductive airway surfactant
comprehensively in terms of PL classes, PC molecular species, surfactant proteins, and surface-tension function. The
first aim of this study was therefore to compare in detail
the PL and PC molecular species composition of tracheal
aspirate samples with that of bronchoalveolar lavage (BAL),
as well as that of surfactant purified from tracheal aspi-
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AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 17 1997
rates (SurfTrachAsp) with those of two fully active surfactant
preparations from BAL (SurfBAL) and the 27,000 3 g pellet
of BAL (SurfP27000). The reasoning for this was that if
these compositions differed significantly, then they might
have different cellular sources. Conversely, if SurfTrachAsp
and surfactant preparations from BAL were similar, this
result would be consistent with the concept that airway
surfactant is derived from overflow of alveolar material.
To assess the activity of conductive airway surfactant as a
potentially physiologically important component of pulmonary surfactant, we included functional analysis of SurfTrachAsp
in comparison with Surf BAL and SurfP27000 in this study.
SP-A, SP-B, and SP-C concentrations were measured in
samples to provide evidence for mechanisms of any differences in surface-tension function between SurfTrachAsp and
alveolar surfactant.
The second aim was to investigate the relationship between the PL compositions of the conductive airway epithelium and those of both tracheal aspirates and SurfTrachAsp.
The cell type-specific composition of membrane PL is determined by a combination of phenotypic expression and
cellular lipid nutrition. Although eicosanoids were described in airways as inflammatory mediators (10, 11), the
molecular compositions of major PL components in airway epithelia as precursors for eicosanoids have never
been determined. Presumably, bronchial and alveolar epithelial cells are exposed to comparable parameters of cellular lipid nutrition derived from the circulation, and any
differences in PL composition will be due to cell-specific
aspects of PL synthesis and turnover. We therefore investigated the biochemical composition of PL classes and
PC molecular species in tracheal aspirates and SurfTrachAsp
of adult pigs in comparison with the underlying epithelium, BAL, surfactant preparations from BAL (Surf BAL,
SurfP27000), and lung parenchyma.
Materials and Methods
Materials
Macroscopically healthy pig lungs were obtained from local slaughter facilities. High-performance liquid chromatography (HPLC)-grade solvents were supplied by Baker
(Deventer, the Netherlands). All other solvents and chemicals were of analytical grade and from various commercial
sources.
Harvesting of Raw Tracheal Aspirates, Bronchoalveolar
Lavage Fluids, and Tissue Specimens
For harvesting tracheal aspirates, the esophagus and heart
were removed from the lungs, and the trachea was cut
open along the membranaceous part. Tracheal mucus was
aspirated by means of a disposable syringe, the trachea
rinsed three times with 2 ml of 154 mmol/liter NaCl, and
the aspirate then added to the original aspirate sample (total volume: 6 to 8 ml). A 6.5-mm blockable catheter was
then placed in the periphery of the left lung and the lower
lobe was lavaged three times with a total volume of 500 ml
of 154 mmol/liter NaCl (recovery: 350 ml). For the removal of cells, BAL fluid was centrifuged for 15 min at
270 3 g and 48C, and the pellet was discarded.
Cell-free BAL fluid was then centrifuged for 3 h at 27,000
3 g and 48C in a Dupont Sorvall Rc-5B centrifuge (Du
Pont De Nemours, Bad Homburg, Germany) to generate
a raw surfactant pellet (P27000; 15 to 25 mmol PL/ml). The
resulting 27,000 3 g supernatant was then recentrifuged at
60,000 3 g for 1 h. The 60,000 3 g supernatant (S60000) was
collected and the 27,000 3 g and 60,000 3 g pellets (P27000,
P60000) were resuspended in 154 mmol/liter NaCl/1.5 mmol/
liter CaCl2 for biochemical and functional analysis (see the
subsequent DISCUSSION). P27000, P60000, and S60000 contained
74.1 6 3.7 mol%, 3.8 6 1.2 mol%, and 22.1 6 2.9 mol% of
the total BAL phospholipids, respectively. Functional
analysis revealed that the active surfactant was harvested
from BAL as P27000, since it reached surface tensions below 5 mN/m in a pulsating bubble surfactometer (Electronetics Co., Amherst, NY) whereas P60000 did not.
For analysis of lung parenchyma, 2 to 3 g of lavaged and
nonlavaged lung parenchyma was cut from the left and
right lower lobe with a pair of scissors. Airway mucosa was
prepared from the underlying tissue at the level of the trachea, carina, and peripheral bronchi, carefully avoiding
any contamination of smaller conductive airway specimens by lung parenchyma (Figure 1). All samples were
stored at 2208C until further analysis. Additional samples
for histology were obtained from the trachea, carina, and
bronchi, using the same procedure. The samples were
fixed in 4% formalin, dehydrated, and embedded in paraffin. Sections were prepared and stained with hematoxylin/
eosin (H&E) for light microscopy.
Surfactant Preparation
Surfactant was prepared from tracheal aspirates, original
BAL, and P27000 according to the method of Shelley (12),
and was designated SurfTrachAsp, SurfBAL, and SurfP27000, respectively. Briefly, a lower phase of 16% NaBr was generated by mixing a 3.75-ml sample with 1.25-ml of 64% NaBr
in 154 mmol/liter NaCl. This solution was overlayered sequentially with 5 ml 13% NaBr (wt/vol) in 154 mmol/liter
NaCl, and then with 2.5 ml of 154 mmol/liter NaCl. After
ultracentrifugation for 1.4 h at 114,000 3 g and 48C, the
surfactant in the samples accumulated as a visible white
band between the 13% NaBr and the 154 mmol/liter NaCl
layers (density 5 1.085 g/ml) (12). The band was aspirated
by means of a disposable syringe, resuspended in doubledistilled water to a total volume of 25 ml, and centrifuged
for 1 h at 27,000 3 g and 48C in a Beckman L8-70 ultracentrifuge using a 70Ti rotor (Beckman Instruments, Mountain View, CA). The pellet was resuspended in 154 mmol/
liter NaCl supplemented with 1.5 mmol/liter CaCl 2 and
stored at 2808C until further analysis. Further centrifugation of the resulting supernatant at 27,000 3 g for 1 h did
not precipitate additional material. Density-gradient residues and supernatants were stored for PL measurements.
Phospholipid Analysis
For PL analysis, lung parenchyma and airway epithelium
were extracted with chloroform/methanol according to the
method of Folch and colleagues (13), and other samples
were extracted according to Bligh and Dyer (14). Lipid extracts were dried under nitrogen and dissolved in chloro-
Bernhard, Haagsman, Tschernig, et al.: Conductive Airway Surfactant Composition and Function
Figure 1. Conductive airway mucosa for PL
analysis. Microphotographs of H&E-stained
sections of the mucosa from: (a) trachea, (b)
carina, and (c) bronchus of pig respiratory
tract. All specimens include the epithelium
(E) and lamina propria (LP). (a and b) Broad
abundance of glandular tissue can be seen.
(c) Smooth-muscle tissue is included. Bar
represents 200 mm.
43
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AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 17 1997
form/methanol (10:1, vol/vol). Quantitation of PL phosphorus was done according to Bartlett (15) after digestion
of the organic compounds at 1908C for 35 min in the presence of 500 ml 70% perchloric acid (wt/vol) and 200 ml
30% hydrogen peroxide (wt/vol). Distribution of total PL
classes was determined by normal-phase HPLC as described previously, using ultraviolet absorption at 205 nm
and subsequent fluorescence emission (excitation wavelength 5 340 nm, emission wavelength 5 460 nm) after
postelution formation of mixed micelles in the presence of
1,6-diphenyl-1,3,5-hexatriene (DPH) (16). The fluorescence response was quantitative for the amount of PL,
whereas the UV response was proportional to the degree
of fatty acyl unsaturation. For the analysis of individual
molecular PC species, a PC fraction was isolated from the
total lipid extract on a 100-mg Varian Bondelut® NH2 disposable cartridge (Varian, Hamburg, Germany). PC molecular species were then resolved by reverse-phase HPLC
and eluted PC species were quantified by postelution fluorescent-derivative formation as outlined earlier (17).
Measurement of Surface Tension
For surface-tension measurements, the PL concentration
of SurfTrachAsp, SurfBAL, or SurfP27000 was adjusted to 4.00,
1.33, and 0.33 mmol/ml, respectively, with 154 mmol/liter
NaCl/1.5 mmol/liter CaCl2. Adsorption, minimal surface
tension (gmin), and maximal surface tension ( gmax) were
then determined with the pulsating bubble surfactometer
(18). Briefly, a bubble was created in a surfactant suspension at 37°C and the static adsorption determined as the
surface tension 10 s after formation of the bubble. The
bubble was then pulsated for 5 min at a frequency of 20 oscillations per min between a minimal bubble radius of 0.4
mm and a maximal radius of 0.55 mm. Adsorption was de-
termined after 10 s, whereas gmin and gmax were calculated,
using the LaPlace equation, after 1, 3, 5, 7, 9, 21, 50, and
100 pulsations.
Protein Analysis
Proteins were quantified according to the method of
Lowry and colleagues (19) in 96-well microtiter plates at
630 nm, using bovine serum albumin (BSA) as a standard.
Sodium dodecylsulfate-polyacrylamide gel electrophoresis
(SDS–PAGE) was done according to Laemmli (20) on a
12.5% polyacrylamide gel with a commercial low-molecular-weight calibration kit (alpha-lactalbumin, 14.4 kD;
trypsin inhibitor, 20.1 kD; carbonic anhydrase, 30 kD;
ovalbumin, 43kD; albumin, 67 kD; phosphorylase b, 94
kD) (Pharmacia-Biotech, Uppsala, Sweden). After overnight fixation in 30% methanol/10% glacial acetic acid/
0.8% formaldehyde, the gels were silver-stained according
to Bloom (21) and stored in 30% methanol. Immunoblot
analysis of SP-A in surfactant samples was done on a 12 %
polyacrylamide gel using a 1:1,000 dilution of a polyclonal
rabbit anti-rat-SP-A antibody and goat anti-rabbit IgG,
conjugated with horseradish peroxidase, as the second antibody. SP-B and SP-C were analyzed from butanol extracts of the surfactant samples. SP-B and SP-C were separated from 200 to 400 nmol PL using a Sephadex LH-60
column (Pharmacia-Biotech), with UV detection of the
proteins at 228 nm, essentially as described previously (22).
Statistics
Statistical analysis was done by one-way analysis of variance (ANOVA) and the two-tailed Student’s t test, using
commercial software (GraphPad InStat Version 1.1, San
Diego, CA). Statistical values were corrected for multigroup comparisons (ANOVA) using the method of Bonferroni.
TABLE 1
Phospholipid composition of tracheal aspirate, BAL, airway epithelium and lung parenchyma
Airway Mucosa
Lung Parenchyma
Phospholipid
Tracheal Aspirate
(mol%; n 5 6)
BAL
(mol%; n 5 3)
Trachea
(mol%; n 5 3)
Carina
(mol%; n 5 6)
Bronchi
(mol%; n 5 6)
Lavaged
(mol%; n 5 4)
Nonlavaged
(n 5 7)
PC
SPH
LPC
PG
PE
PI
PS
(UV/FL)pc
Total PL
73.6 6 2.3†
3.5 6 0.9*
1.4 6 0.7
3.5 6 0.8*
13.7 6 1.3‡
4.3 6 1.0
Trace
0.24 6 0.03
1.10 6 0.20
85.4 6 2.1
0.7 6 0.2
Trace
6.1 6 0.8
4.8 6 1.0
3.0 6 0.4
Trace
0.20 6 0.01
0.23 6 0.03 (mmol/ml)
45.2 6 1.4
8.5 6 0.2§
0.5 6 0.1
3.4 6 1.5
39.5 6 0.9§
2.5 6 0.4
0.4 6 0.4
0.79 6 0.03¶
8.7 6 1.7§§
44.4 6 0.5
8.9 6 0.4§
0.7 6 0.1
0.3 6 0.1§
39.4 6 0.5‡
4.8 6 0.4§
1.6 6 0.5§
0.81 6 0.04**
11.0 6 1.4¶¶
47.1 6 0.5
8.0 6 0.4§
0.7 6 0.1
0.3 6 0.1§
39.2 6 0.4¶
4.6 6 0.3§
0.1 6 0.1
0.73 6 0.03**
11.2 6 0.4¶¶
50.1 6 0.9
13.4 6 0.4
0.6 6 0.2
3.3 6 0.7
30.4 6 0.8
2.2 6 0.6
Trace
0.43 6 0.02
10.3 6 0.62
56.2 6 1.7
10.6 6 1.1
0.6 6 0.2
3.7 6 0.6
26.7 6 0.9
2.2 6 0.4
Trace
0.39 6 0.01
27.1 6 1.14 (mmol/g)
Tissue, tracheal aspirate, and lavage samples were extracted with chloroform/methanol. Total phospholipid was measured as phospholipid phosphorus, and phospholipid classes were analyzed with HPLC. Data are presented as mean 6 SE.
Definition of abbreviations: n 5 number of samples analyzed. Concentraction data are expressed as mmol/ml (fluid samples) and mmol/g wet weight (tissue samples).
*P , 0.05 versus BAL.
†
P , 0.01 versus BAL.
‡
P , 0.001 versus BAL.
§
P , 0.05 versus lavaged lung parenchyma.
¶
P , 0.01 versus lavaged lung parenchyma.
**P , 0.001 versus lavaged lung parenchyma.
§§
P , 0.01 versus nonlavaged lung parenchyma.
¶¶
P , 0.001 versus nonlavaged lung parenchyma.
Bernhard, Haagsman, Tschernig, et al.: Conductive Airway Surfactant Composition and Function
TABLE 2
Phospholipid composition of surfactant preparations
and BAL fractions
Phospholipid
PC
SPH
LPC
PG
PE
PI
PS
(UV/FL)PC
SurfTrachAsp
(mol%; n 5 4)
SurfBAL
(mol%; n 5 11)
SurfP27000
(mol%; n 5 12)
P27000
(mol%; n 5 12)
88.5 6 0.7
0.5 6 0.1
0.2 6 0.2
6.6 6 0.5
2.2 6 0.4
1.9 6 0.2
Trace
0.20 6 0.02
85.6 6 1.8
1.0 6 0.5
Trace
8.6 6 0.7
2.9 6 0.8
2.0 6 0.2
Trace
0.13 6 0.02
87.9 6 0.7
0.5 6 0.1
Trace
8.3 6 0.6
1.9 6 0.2
1.4 6 0.3
Trace
0.14 6 0.01
86.0 6 0.8
0.8 6 0.1
Trace
7.9 6 0.6
2.8 6 0.3
2.5 6 0.3
Trace
0.16 6 0.01
Lipid extracts were dissolved in chloroform:methanol 1:4 (vol/vol) and separated for PL classes by HPLC. Data are presented as means 6 SE.
Definition of abbreviation: n 5 number of experiments.
Results
Amount of Phospholipids
The PL concentration was substantially lower in airway
mucosa (8.7 6 1.7, 11.0 6 1.4, and 11.2 6 0.4 mmol/g in trachea, carina, and bronchi, respectively) than in nonlavaged
lung parenchyma (27.10 6 1.36 mmol/g, P , 0.001) (Table
1). Representative sections of the airway mucosa samples
are shown in Figure 1. Tracheal aspirates and BAL samples contained 1.10 6 0.17 and 0.23 6 0.03 mmol PL/ml, respectively (Table 1). Recovery of SurfTrachAsp by densitygradient centrifugation was 11.6 6 4.8 mol% of the total
PL in tracheal aspirates. By comparison, recoveries of
SurfBAL from BAL and of Surf P27000 from the highly concentrated P27000 were 36.4 6 5.9 mol% and 92.2 6 1.4
mol%, respectively. SurfP27000 contained 68.3 6 5.2% of total BAL PL.
45
Composition of Phospholipid/Classes
The PL composition of tracheal aspirate samples was similar to that of BAL and lung parenchyma samples, but not
to that of the underlying epithelium (Table 1). Airway epithelium contained less PC and, with the exception of tracheal epithelium, apparently no phosphatidylglycerol (PG).
In contrast, the phosphatidylethanolamine (PE) concentration was increased in airway epithelium as compared
with both lavaged and nonlavaged lung parenchyma (Table 1). Moreover, the high UV-to-fluorescence ratio (16)
indicated an increased concentration of unsaturated fatty
acids in PC from airway epithelium, as compared with the
highly saturated PC composition of tracheal aspirates,
BAL, and lung parenchyma (Table 1).
The concentrations of sphingomyelin (SPH), PE, and
phosphatidylinositol (PI) were higher in tracheal aspirates
than in BAL fluid (Table 1). In contrast, SurfTrachAsp had the
same composition of PL classes as the alveolar surfactant
preparations SurfBAL and SurfP27000 (Table 2). Although the
UV-to-fluorescence ratio of PC was higher in SurfTrachAsp
than in SurfBAL and SurfP27000 (Table 2; P , 0.05), the PC
composition of SurfTrachAsp was nevertheless more similar
to that of SurfBAL and SurfP27000 than to that of the underlying airway epithelium. Therefore, we subsequently reinvestigated our samples for PC molecular species.
Composition of Phosphatidylcholine Molecular Species
The major PC species present in tracheal aspirates were
dipalmitoyl-PC (PC16:0/16:0), palmitoyloleoyl-PC (PC16:
0/18:1), palmitoylmyristoyl-PC (PC16:0/14:0), palmitoylpalmitoleoyl-PC (PC16:0/16:1), and palmitoyllinoleoyl-PC
(PC16:0/18:2) (Table 3). This composition was similar but
not identical to the PC molecular species of BAL fluid (Table
3). PC16:0/16:0 was the predominant PC molecular species
in tracheal aspirates (49.6 6 0.8%), BAL (56.2 6 1.2%),
TABLE 3
Molecular species of phosphatidylcholine in tracheal aspirate, BAL,
airway epithelium and lung parenchyma
Airway Mucosa
Lung Parenchyma
PC Species
Raw Tracheal
Aspirate
(mol%; n 5 6)
BAL
(mol%; n 5 3)
Trachea
(mol%; n 5 3)
Carina
(mol%; n 5 6)
Bronchi
(mol%; n 5 6)
Lavaged
(mol%; n 5 8)
Nonlavaged
(n 5 8)
16:0/14:0
16:0/16:0
16:0/16:1
16:0/18:1
16:0/18:2
18:0/18:2
16:0/20:4
16:0/22:6
18:0/20:4
18:1/18:1
18:1/18.2
Others
9.7 6 0.5
49.6 6 0.8*
8.1 6 0.5
14.4 6 0.8*
6.5 6 0.2
1.5 6 0.3
1.9 6 0.2
0.9 6 0.1
0.8 6 0.1
1.4 6 0.2
1.6 6 0.1
3.7 6 0.4
9.2 6 0.4
56.2 6 1.2
7.5 6 0.4
10.0 6 0.6
6.0 6 0.7
1.8 6 0.2
1.6 6 0.4
0.4 6 0.1
0.7 6 0.2
0.8 6 0.2
1.2 6 0.1
4.6 6 0.5
1.0 6 0.2§
8.5 6 0.4§
1.6 6 0.2†
25.2 6 0.8‡
12.6 6 2.2†
10.9 6 0.6§
5.8 6 0.2‡
2.2 6 0.2†
6.5 6 0.3
2.3 6 0.3
4.3 6 0.3‡
19.9 6 1.1
0.9 6 0.2§
8.6 6 0.3§
1.3 6 0.1§
26.1 6 0.5§
13.5 6 0.9§
10.5 6 0.5§
6.4 6 0.3§
2.1 6 0.1†
5.8 6 1.1
1.8 6 0.2
3.9 6 0.3‡
20.1 6 0.9
1.0 6 0.2§
8.7 6 0.6§
1.2 6 0.2§
25.7 6 1.2§
10.1 6 0.8
9.1 6 1.0§
4.6 6 0.4
2.9 6 0.6§
6.9 6 0.6†
4.0 6 0.5§
4.2 6 0.6‡
22.7 6 1.0†
3.0 6 0.3
33.8 6 1.0
3.7 6 0.3
19.0 6 0.6
7.8 6 0.4
3.7 6 0.3
3.8 6 0.2
0.9 6 0.1
4.3 6 0.2
1.0 6 0.1
2.2 6 0.1
17.0 6 1.1
5.4 6 0.3§
42.5 6 1.6§
4.7 6 0.1
16.3 6 0.7†
6.6 6 0.3
2.8 6 0.1
2.8 6 0.1†
1.2 6 0.1
3.1 6 0.1
0.5 6 0.1
1.5 6 0.1
12.6 6 1.2†
PC molecular species were purified on aminopropyl BondElut cartridges and resolved by reverse phase HPLC. Data are means 6 SE.
Definition of abbreviation: n 5 number of samples.
*P , 0.05 versus BAL.
†
P , 0.05 versus lavaged lung parenchyma.
‡
P , 0.01 versus lavaged lung parenchyma.
§
P , 0.001 versus lavaged lung parenchyma.
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AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 17 1997
and lavaged lung parenchyma samples (32.7 6 1.2%). This
value was significantly lower in tracheal aspirates than in
BAL samples (P , 0.001). In contrast, the PC16:0/16:0
content of airway mucosa was considerably lower. Mucosal preparations from trachea, carina, and bronchi, containing both surface epithelium and glandular tissue with
minor contaminations from connective tissue (Figure 1),
had essentially identical PC compositions, with PC16:0/
16:0 contributing consistently less than 10% (Table 3). The
major species present in airway mucosa were PC16:0/18:1,
PC16:0/18:2, and stearoyllinoleoyl PC (PC18:0/18:2). In
addition, airway mucosa contained more palmitoylarachidonoyl PC (PC16:0/20:4), stearoylarachidonoyl PC (PC18:
0/20:4), and palmitoyldocosahexaenoyl PC (PC16:0/22:6)
than did tracheal aspirates, BAL, and lung parenchyma.
Surfactant isolated from tracheal aspirates and BAL
(SurfTrachAsp, SurfBAL, and Surf P27000) contained, respectively, 59.7 6 1.1, 62.9 6 1.0, and 63.2 6 1.8 mol% PC16:0/
16:0, and there were only minor differences in the concentrations of other PC species (Table 4).
Surface Activity of SurfTrachAsp, SurfBAL, and SurfP27000
To correlate the lipid biochemical findings with functional
data, we measured static adsorption, gmin, and gmax of
SurfTrachAsp, SurfBAL, and SurfP27000. Static adsorption of
SurfTrachAsp was much slower than that of Surf BAL and
SurfP27000 at all concentrations measured (0.33, 1.33, and
4.00 mmol PL/ml, respectively), resulting in higher surfacetension values after 10 s (Table 5). At these PL concentrations, gmin after 5 min of cycling was 24.6 6 2.0 mN/m,
21.8 6 0.5 mN/m, and 18.2 6 1.1 mN/m, respectively (Figure 2a). In no analysis of SurfTrachAsp did gmin fall to a value
below 5 mN/m. At the PL concentrations evaluated, gmax
initially ranged from 56.6 6 1.3 to 69.4 6 1.6 mN/m (Figure 2b). Values of 35 to 40 mN/m, which are characteristic
of alveolar surfactant preparations, were not achieved
TABLE 4
Molecular species of phosphatidylcholine in surfactant
preparations and BAL fractions
PC Species
SurfTrachAsp
(mol%; n 5 4)
SurfBAL
(mol%; n 5 11)
SurfP27000
(mol%; n 5 12)
P27000
(mol%; n 5 12)
16:0/14:0
16:0/16:0
16:0/16:1
16:0/18:1
16:0/18:2
18:0/18:2
16:0/20:4
16:0/22:6
18:0/20:4
18:1/18:1
18:1/18:2
Others
6.4 6 0.2
59.7 6 1.1
5.1 6 0.3
8.5 6 0.6
9.1 6 0.4
2.4 6 0.4
0.1 6 0.1
0.5 6 0.1
0.7 6 0.3
1.6 6 0.7
1.8 6 0.2
4.1 6 1.1
9.2 6 0.8
62.9 6 1.0
7.1 6 0.4
9.7 6 0.9
5.3 6 0.7
0.9 6 0.2*
0.2 6 0.1
0.0 6 0.0‡
0.1 6 0.1*
0.9 6 0.3
0.7 6 0.1†
3.0 6 0.4
9.1 6 1.2
63.2 6 1.8
6.9 6 0.5
9.9 6 1.2
6.3 6 0.7
1.1 6 0.3
0.1 6 0.1
0.0 6 0.0‡
0.0 6 0.0†
0.9 6 0.1
0.6 6 0.2†
3.5 6 0.3
9.7 6 1.0
60.2 6 1.4
7.0 6 0.5
10.5 6 1.1
6.3 6 0.9
1.5 6 0.3
0.2 6 0.1
0.0 6 0.0‡
0.1 6 0.1*
0.5 6 0.2
1.0 6 0.1*
2.9 6 0.5
PC molecular species were purified using aminopropyl BondElut cartridges
and resolved by reverse phase HPLC. Data are means 6 SE.
Definition of abbreviation: n 5 number of samples.
*P , 0.05 versus SurfTrachAsp.
†
P , 0.01 versus SurfTrachAsp.
‡
P , 0.001 versus SurfTrachAsp.
even at high PL concentrations (4 mmol/ml). Nevertheless,
surface tension after static adsorption for 10 s, gmin during
the first bubble pulsation, and the number of pulsations
necessary for achieving stable gmin values all remained concentration-dependent with SurfTrachAsp (Table 5, Figure 2a).
These characteristics of conductive airway surfactant
function were compared with those of alveolar surfactant
preparations of the same lungs. In contrast to SurfTrachAsp,
SurfBAL and SurfP27000 showed rapid adsorption (Table 5),
gmin values below 5 mN/m ( Figures 2c and 2e), and gmax
values around 30 to 40 mN/m (Figures 2d and 2f). All of
these parameters were concentration-dependent, and were
identical for both alveolar surfactant preparations (Figures 2c through 2f). Even at a concentration of 0.33 mmol
PL/ml, minimal surface tension was 15.4 6 5.3 mN/m and
15.9 6 2.8 mN/m for SurfBAL and SurfP27000, respectively.
Surfactant Protein Analysis
Since the composition of PL classes or PC molecular species did not offer any explanation for the functional differences between SurfTrachAsp and either SurfBAL or SurfP27000,
we analyzed the various surfactant preparations for protein concentration and composition. The protein concentration of SurfTrachAsp (0.28 6 0.05 mg protein/mmol PL,
n 5 13) was significantly higher (P , 0.001) than that of
either SurfBAL (0.11 6 0.01 mg protein/mmol PL, n 5 4)
or SurfP27000 (0.10 6 0.01 mg protein/mmol PL, n 5 11).
SDS-PAGE analysis (Figure 3a) confirmed that SurfBAL,
SurfP27000, and P27000 all contained two protein bands at 32
to 36 kD, which is the molecular weight range of glycosylated surfactant apoprotein A monomers (1), whereas
P60000 and S60000 (see MATERIALS AND METHODS) were
not. Although these protein bands of 32 to 36 kD were
also present in SurfTrachAsp, other protein bands were more
prominent in these preparations than in Surf BAL and
SurfP27000. We therefore determined SP-A semiquantitatively in SurfTrachAsp as compared with SurfP27000 by immunoblot analysis, applying identical amounts of PL (35
nmol) per lane to a 12% polyacrylamide gel. This result
(Figure 3b) demonstrated clearly that SP-A is considerably decreased in Surf TrachAsp as compared with alveolar
surfactant (SurfP27000). Moreover, since surface adsorption
strongly depends on the presence of SP-B and SP-C, we
analyzed these proteins in SurfP27000 and SurfTrachAsp using
Sephadex LH 60 gel filtration (Figure 4). SurfP27000 (n 5 3)
contained 10.8 6 1.0 mg SP-B and 16.2 6 3.6 mg SP-C/
mmol PL. These values are comparable with the respective
concentrations in surfactant from BAL of pig, dog, and
sheep (data not shown). By contrast, Surf TrachAsp (n 5 3
from two to five pooled samples each) did not contain
measureable amounts of SP-B and SP-C (Figure 4).
Discussion
Our analyses of material from adult porcine lungs showed
that the composition of both PL classes and, particularly,
PC molecular species in tracheal aspirate samples, were
similar to those in BAL, and were markedly different from
the PL and PC composition of airway epithelium. The PC
of airway mucosa was characteristic of the molecular composition of mucosa from other tissues (23), whereas the
Bernhard, Haagsman, Tschernig, et al.: Conductive Airway Surfactant Composition and Function
47
Figure 2. Surface activity of
SurfTrachAsp in comparison with
alveolar surfactant preparations.
(a, c, e) Minimal (closed symbols) and (b, d, f) maximal (open
symbols) surface tensions of
SurfTrachAsp (a, b), SurfBAL (c, d)
and SurfP27000 (e, f) determined at
0.33 (squares), 1.33 (triangles),
and 4.00 (circles) mmol PL/ml
as described in Materials and
Methods. Data are means 6 SE
of four to six experiments. If no
error bar is shown, SE was
smaller than the symbol.
tracheal aspirate PC composition was characteristic of lung
surfactant (1). The higher content of SPH, PE, and PI in
initial tracheal aspirate samples as compared with BAL
suggests either a significant contribution of cell membrane
material or secretion of material enriched in these phospholipids from the airway epithelium. It is not possible to
TABLE 5
Static adsorption of SurfTrachAsp in comparison to
SurfBAL and SurfP27000
Surface Tension (mN/m)
Phospholipid
Concentration
SurfTrachAsp
(n 5 6)
SurfBAL
(n 5 6)
SurfP27000
(n 5 6)
0.33 mmol/ml
1.33 mmol/ml
4.00 mmol/ml
62.5 6 2.4
50.1 6 3.7
47.5 6 2.4
52.5 6 8.2
27.5 6 2.0†
27.1 6 1.2†
54.8 6 3.4
31.7 6 5.3*
25.1 6 0.4†
Surface tension was determined after 10 s of static adsorption as indicated in
Materials and Methods. Data are presented as means 6 SE (mN/m).
Definition of abbreviation: n 5 number of samples.
*P , 0.05 versus SurfTrachAsp.
†
P , 0.01 versus SurfTrachAsp.
distinguish between these putative processes, as little evidence has been presented to support active PL secretion in
the airways (24–26). Although the trachea may also contribute some PG to the airway mucus (27), smaller airway
epithelia do not contain PG. Since airway mucus and its
constituents are obviously transported from more distal
parts of the airways by mucociliary activity of the epithelium, PG secretion by the tracheal mucosa is unlikely to be
a major contributor to conductive airway surfactant. Moreover, conductive airway surfactant isolated from tracheal
aspirate samples by sodium bromide density-gradient centrifugation (SurfTrachAsp) (12) exhibited a PL and PC molecular species composition essentially identical to those of
BAL and of surfactant prepared from BAL (SurfBAL and
SurfP27000). This result strongly suggests that, whatever
their cellular source, the increased contents of SPH, PE,
and PI in initial tracheal aspirate samples were not integral
components of conductive airway surfactant (SurfTrachAsp).
The simplest explanation for our results is that SurfTrachAsp
is derived from alveolar material rather than being secreted by airway epithelial or glandular tissue. Although it
is evidently not possible to provide conclusive proof of this
hypothesis solely by compositional analysis, it is supported
48
AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 17 1997
Figure 3. SDS–PAGE and immunoblot analysis of SP-A in
SurfTrachAsp and Surf P27000 preparations. SDS-PAGE was done
with (a, b) unstained and (b) prestained molecular weight markers. (a) Separation of the indicated surfactant preparations was
done on a 12% polyacrylamide gel and the proteins (5 mg/lane)
were silver-stained, as indicated in Materials and Methods. The
arrow indicates the range of 32 to 36 kD, representing glycosylated surfactant apoprotein A monomer. For abbreviations see
text. (b) Blotting was done with a polyclonal rabbit-anti-rat-SP-A
antibody and subseqent incubation with horseradish peroxidaseconjugated goat antirabbit antibody. Lane 1: prestained molecular weight standards; Lane 2: 1 mg porcine SP-A; Lanes 3, 5, 7:
SurfTrachAsp samples (35 nmol PL applied to gel); Lanes 4, 6, 8:
SurfP27000 samples (35 nmol PL applied to gel); Lane 9: 0.1 mg porcine SP-A; Lane 10: unstained molecular weight standards.
by considerable circumstantial evidence. Airway mucosa
samples, consisting of surface epithelium together with
glandular tissue and minor contaminations by connective
tissue, contained only half the concentration of total PL,
only minor amounts of PC16:0/16:0 and, with the exception of tracheal epithelium, no PG as compared with lung
parenchyma. Instead of the high concentration of PC16:0/
16:0 measured in all the various surfactant fractions including SurfTrachAsp, airway mucosal PC was enriched in
the mono- and diunsaturated species PC16:0/18:1, PC16:0/
18:2, and PC18:0/18:2, together with the highly unsaturated species PC16:0/20:4, PC18:0/20:4, and PC16:0/22:6.
Consequently, the overall PL content of airway mucosa is
unlikely to have been the source of surfactant enriched in
PC16:0/16:0, although it remains a potential substrate for
Figure 4. Representative HPLC analyses of SP-B and SP-C in
SurfTrachAsp compared with Surf P27000. Surfactant was prepared
from tracheal aspirates (Surf TrachAsp) and the 27,000 3 g pellet
of BAL fluid (Surf P27000). The samples were extracted with
n-butanol and analyzed for SP-B and SP-C as described in Materials and Methods. (a) SurfP27000. (b) SurfTrachAsp. PL 5 phospholipid.
release of the eicosanoid precursor arachidonic acid (10,
28, 29). It is, however, possible that a specialized organelle
or cell type within the airways may be involved in secretion of such surfactant material. Although small amounts
of PL material may be present in the granules and secretions of glandular epithelial cells, specific secretion of
PC16:0/16:0 and PG from such cells has never been demonstrated (26, 30). However, since no detailed analyses
have been made of the PC molecular compositions of either individual cell types or isolated cellular subfractions,
neither possibility can be assessed directly.
Comparison with other cell types that actively secrete
PL, such as hepatocytes, type II alveolar cells, and gastric
mucosal cells may prove useful in this regard. PL is secreted by hepatocytes both in bile and in lipoprotein complexes (31, 32), and by the gastric mucosa to form a protective hydrophobic barrier (23, 33). In all cases, although a
degree of molecular species selectivity has been demonstrated in these regulated secretion processes, the PC species secreted were always also major components of the
total cellular PL extract. Airway mucosal PL classes and
PC molecular species compositions were, however, directly comparable with those in our previous report of the
composition of the gastric mucosa (23), which secretes
principally PC16:0/18:1 and PC16:0/18:2 rather than PC16:
0/16:0. In contrast, although secreted alveolar surfactant is
enriched in PC16:0/16:0 as compared with lung tissue PC,
PC16:0/16:0 remains a major component of PC in this tissue. Because PC16:0/16:0 contributed only 9% to airway
Bernhard, Haagsman, Tschernig, et al.: Conductive Airway Surfactant Composition and Function
mucosal PC, rather than the more than 30% in lavaged
lung tissue, this indirect evidence suggests that secretion of
PL enriched in PC16:0/16:0 from a putative specialized organelle is unlikely. In support of this view is the lack of
lamellar bodies characteristic of surfacant secretion in the
airway epithelium and glandular tissue (26, 34). Moreover,
significant contributions of a specialized airway cell type
to the PC16:0/16:0 content of conductive airway surfactant
would imply increased synthesis and storage of PC16:0/
16:0, which should be detectable by labeling in vivo with
radioactive precursors, followed by autoradiography. Although such a specific study has not been performed, the
early experiments of Chevalier and Collet (35) that confirmed the type II cell as the cellular source of alveolar
surfactant did not report any such cell in conductive airways.
The similarity of both PL classes and PC molecular species compositions of airway mucosa to those of gastric mucosa rather than lung parenchyma suggests that aspects of
the metabolism of lipid mediators may be similar in conductive airways and the gastric mucosa. PC from both tissues contains significant amounts of highly unsaturated
fatty acids. The present study has determined, for the first
time in terms of individual PC molecular species, the PC
composition of conductive airway mucosa, and has demonstrated concentrations of PC16:0/20:4 and PC18:0/20:4
identical to gastric mucosal PC (23). Docosahexaenoic
acid was additionally present as PC16:0/22:6. These data
support the concept that airway epithelia are potential
sources for release of the eicosanoid precursor arachidonic
acid from PC16:0/20:4 and PC18:0/20:4 (10, 28, 29). Moreover, in addition to cellular PC16:0/20:4 and PC18:0/20:4,
the minor amounts of these molecules present in airway
mucus may serve as a source for eicosanoid or other mediator synthesis by resident or invading inflammatory cells.
Conductive airway surfactant may be important for mucociliary clearance (2, 4, 6, 36–40), and an impaired surface-tension function of this material has been associated
with pathologic mucociliary clearance in chronic suppurative airway inflammation (41, 42). However, although the
PL and PC compositions of conductive airway surfactant
(SurfTrachAsp) were identical to those of original BAL,
P27000, SurfBAL, and SurfP27000, the adsorption velocity and
minimal surface tension (gmin) of SurfTrachAsp from the
healthy porcine lungs in the present study were impaired
as compared with those of SurfBAL and SurfP27000. This is in
contrast to the situation in full-term newborn infants, in
which highly active surfactant can be isolated from pharyngeal aspirates through the same technique as used in
our study (43, 44). However, the perinatal period is a different physiologic setting, since at birth considerable
amounts of surfactant are squeezed out from the lung periphery rather than transported by mucociliary clearance.
Possible explanations for the differences in surface-tension function in SurfTrachAsp from adult lungs as compared
with alveolar surfactant are that the surface pressure of
the surfactant layer at the alveolar air–liquid interface
leads to a preferential squeezing out of individual surfactant components from that interface, and that fully active
surfactant does not enter the airways. Moreover, surfactant proteins may be retained or degraded in the periphery
49
of the lungs. Electron microscopic studies of adult lungs
have shown that the surfactant structures present in the sol
phase of the airway mucus are composed primarily of
small unilamellar rather than large multilamellar vesicles
(2). Within the alveolus, such a distribution would imply a
preponderance of surface-inactive surfactant destined for
recycling by the type II alveolar epithelial cell (1, 45, 46).
Assuming a comparable structure–function relationship in
the airways, this could suggest that adult conductive airway surfactant is derived from surface-inactive alveolar
surfactant. Our data support this concept, since SurfTrachAsp
shows a different protein composition in terms of decreased
SP-A levels and the absence of measurable amounts of SP-B
and SP-C. Additionally, the lower surface activity and
unilamellar structure of conductive airway surfactant may
be due to the presence of inhibitory proteins derived from
airway secretions, since the total protein concentration was
higher in SurfTrachAsp than in SurfBAL and SurfP27000. This is
in accord with earlier reports on the tight association of
surfactant to mucus components (40, 47).
In summary, the biochemical results of our study suggest that the mucosal cells of larger conductive airways of
adults are probably not major sources of newly secreted
intact surfactant particles, although they probably contribute individual PL components to conductive airway secretions. The substantial amounts of PL and PC molecular
species recovered from surfactant preparations of airway
secretions may have been derived from alveolar surfactant, as suggested by their identical compositions and by
the completely different PL and PC composition of the underlying airway mucosa. Nevertheless, final proof of this
hypothesis can only be achieved with additional studies,
such as with isolated tracheal preparations. The surfacetension function of the conductive airway surfactant was
substantially impaired in comparison with alveolar surfactant, even after purification by density-gradient centrifugation, and the concentrations of surfactant proteins SPA, SP-B, and SP-C were decreased. Tracheal aspirate samples from adult subjects may be an accessible source of
conductive airway surfactant (SurfTrachAsp) with a PL composition comparable to that of alveolar surfactant (SurfBAL, SurfP27000). However, although such samples may
prove useful for assessing the functional adequacy of conductive airway surfactant, they are unlikely to provide results representative of alveolar surfactant function and
protein composition.
Acknowledgments: The authors thankfully acknowledge the excellent technical
assistance of Ch. Acevedo and K. Westermann. This study was supported by
Deutsche Forschungsgemeinschaft Grant HA 1959/2-1.
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