1 Children's Hospital Medical Center, Division of Pulmonary Biology, Cincinnati, Ohio 45229-3039; and 2 Byk Gulden, D-76403 Constance, Germany
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ABSTRACT |
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The developing lung contains surfactant protein (SP) C mRNA levels comparable to term values before mature type II cells and alveolar surfactant lipids are detectable. Estimates of the amount of mature SP-C in the alveolar lavages of preterm lungs are not available. We used an antibody to a recombinant human SP-C to measure the amount of SP-C in alveolar lavages of preterm fetal rabbits, ventilated preterm rabbits, and term rabbits. The amounts of SP-C were compared with the amounts of saturated phosphatidylcholine (Sat PC). Median Sat PC amounts increased about 680-fold, and median SP-C values increased by over 5,000-fold in alveolar washes from 27 days gestation to term. There was no increase in Sat PC or SP-C with ventilation at 27 and 28 days gestation, but ventilation increased both Sat PC and SP-C at 29 days gestation. The molar percent of SP-C relative to Sat PC also increased with gestational age and with ventilation at 29 days gestation. proSP-C was abundant in a membrane fraction from lung tissue at 27 and 28 days gestation when minimal mature SP-C was detected in alveolar washes. At 29 days and at term, proSP-C decreased in membrane fractions. The preterm lung that is surfactant lipid deficient is also severely deficient in mature SP-C.
lung immaturity; respiratory distress syndrome; prematurity; phosphatidylcholine
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INTRODUCTION |
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SURFACTANT PROTEIN (SP) C is an extremely hydrophobic 35-amino acid protein that is only found in the lung (4). SP-C augments the surface tension, lowering properties of surfactant phospholipids by accelerating adsorption and surface spreading (23). Surfactants that contain only SP-C or analogs of SP-C are similar to native surfactants that also contain SP-A and SP-B when tested in surfactant-deficient preterm lungs or saline-lavaged adult lungs (7, 10). The mRNA for SP-C is present in the distal epithelium of the branching airways of human and animal lungs before midgestation (9, 14, 25). The unprocessed proSP-C (Mr 21,000) also can be found by immunocytochemistry in human fetal lungs by 15 wk gestation (14). Although in the developing rabbit lung SP-C mRNA levels are easily detectable by Northern blots at 19 days gestation and increase to term levels by 24 days (16), transcription rate is low until about 28 days as assessed by nuclear run-on assays (6). proSP-C is processed in the trans-Golgi, and de novo synthesized mature SP-C is in lamellar bodies in type II cells (5, 22). Mature SP-C is thought to be cosecreted to the alveoli with the surfactant phospholipids and SP-B (24). Although chromatographic and chemical methods for the measurement of SP-C have been reported (18, 21), they have not been used generally and are not useful for detecting small amounts of SP-C. Despite multiple attempts, antibodies have not been raised to native SP-C, but an antibody to a recombinant human SP-C analog that cross-reacts with mature SP-C is now available. There is no information about the amount of mature SP-C in the developing lung. Therefore, we have used Western blots to quantify mature SP-C in alveolar washes and to identify proSP-C in the lung tissue of preterm fetal and preterm ventilated rabbits in comparison to term rabbits.
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METHODS |
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Delivery and ventilation of rabbits. Time-mated New Zealand White rabbits at 27 days gestation (n = 6), 28 days gestation (n = 4), and 29 days gestation (n = 3) were lightly anesthetized with intravascular pentobarbital sodium followed by spinal anesthesia using 2 ml of 2% lidocaine and 0.5% bupivacaine (1:1 vol/vol). The does received oxygen by face mask as the preterm rabbits were sequentially delivered by cesarean section. One-half of each litter was killed with intratracheal lidocaine before breathing and were the unventilated fetal rabbits. The other half of each litter was ventilated (19). The newborns to be ventilated were given 0.1 mg/kg acepromazine and 10 mg/kg ketamine by intraperitoneal injection. Tracheotomy was performed, and a tube made from an 18-gauge stainless steel needle was secured in the trachea of each rabbit. The initial lung inflation was achieved by ventilating each rabbit with five breaths of 100% oxygen with an anesthesia bag using just enough pressure to visibly move the chest. The rabbits were then transferred to a 37°C temperature-controlled ventilator-plethysmography system set to initially deliver a peak inspiratory pressure of 35 cmH2O; rate 30 breaths/min; inspiratory time 1 s; 100% oxygen; and 3 cmH2O positive end-expiratory pressure. The peak inspiratory pressures were individually adjusted for each rabbit during the 15-min ventilation period to achieve tidal volumes of 7-8 ml/kg as measured with a pneumotachometer. These tidal volumes were previously shown to achieve physiological PCO2 values (19). The other ventilator settings were not changed during the ventilation. Compliance was calculated as tidal volume per kilogram divided by the peak inspiratory pressure minus positive end-expiratory pressure measured after 15 min of ventilation. At the end of 15 min of ventilation, the endotracheal tube was obstructed, and the rabbits were killed with lidocaine intrathecally.
Alveolar washes and processing of lungs. The nonventilated and ventilated preterm rabbits, seven 1-day-old term newborn rabbits, and several of the does had standardized lung lavages using 5 aliquots of 0.9% NaCl sufficient to fully inflate the lungs of the open-chested animals (19). Each saline lavage was flushed in and out of the lungs three times, and the five lavages were pooled. The lavages from three to six rabbits at 27 days gestation, two rabbits at 28 days gestation, and one to three rabbits at 29 days gestation were combined for subsequent processing. The pooled alveolar lavages were centrifuged at 40,000 g for 30 min at 4°C to recover the sedimentable surfactant as a pellet referred to as the heavy- or large-aggregate surfactant fraction (27). The lung tissue after alveolar lavage was weighed and homogenized in saline followed by low-speed centrifugation at 1,000 g for 5 min at 4°C. A membrane fraction then was recovered as a pellet after centrifugation of the supernatant of the low-speed spin at 10,000 g for 20 min at 4°C. The large-aggregate surfactant and lung membranes were resuspended in saline, and an aliquot was used to measure saturated phosphatidylcholine (Sat PC) by assay of phosphorus after chloroform-methanol extraction, osmium tetroxide treatment of the lipid extracts, and alumina column chromatography (2, 15).
Anti-SP-C antibody. The anti-SP-C antibody was raised in rabbits using a human recombinant SP-C with the human 34-amino acid sequence altered by replacing the cysteines in positions 4 and 5 with phenylalanine and methionine and in position 32 with isoleucine (10). These substitutions improve the solubility and prevent the aggregation and denaturation of the peptide, which has equivalent clearance kinetics from the air spaces and lungs of rabbits and preterm lambs as native SP-C (11, 12)
Western blots for SP-C. SDS-polyacrylamide gel electrophoresis of large-aggregate surfactant and lung membrane samples was carried out using 10-20% gradient Tricine gels (Novex, San Diego, CA). Samples containing 1-5 µg of Sat PC were electrophoresed along with 5 ng of recombinant human SP-C as standard. Proteins were transferred to polyvinylidene difluoride (PVDF) paper (Bio-Rad, Richmond, CA) for immunoblot analysis using rabbit anti-recombinant SP-C serum (Byk Gulden). The PVDF membrane was first blocked with 5% BSA (wt /vol) in Tris-buffered saline, pH 7.4, containing 0.1% (vol/vol) Tween 20 (TBS-T). The rabbit anti-SP-C serum was diluted (1:25,000) in TBS-T and incubated with the PVDF paper overnight. Horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin (Calbiochem, La Jolla, CA) diluted 1:10,000 in TBS-T was used as secondary antibody. The immunoblots were developed using enhanced chemiluminescence substrates (Amersham, Arlington Heights, IL). Estimates of the amount of SP-C in different samples were made by calculating the relative band densities using Alpha-Imager 2000 Documentation and Analysis Software (Alpha Innotech, San Leandro, CA). The relative densitometric values obtained were within a linear range dependent on the sample amounts loaded and the film exposure time. Reaction of higher-molecular-weight rabbit immunoglobulin bands that were present in the samples did not complicate analysis of the specific 5-kDa mature SP-C band.
Data analyses.
Values for the physiological measurements (tidal volumes, compliances)
are given as means ± SE, with n
equaling the number of animals (Table 1).
Values for Sat PC and SP-C are given as medians because the
measurements of the pooled groups include values at the limits of
detection of the assays at the early gestational ages and distributions
of values that are not normal. Significance was tested by the
nonparametric Mann-Whitney procedure.
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RESULTS |
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The tidal volumes per kilogram were similar for the rabbits ventilated
at each gestation (Table 1), and compliance values were the same for
27- and 28-day- gestation rabbits, but compliance for 29-day rabbits
increased (P < 0.01 vs. 27 and 28 days gestation). The fetal rabbits at 27 days gestation had only 0.15%
of the 13.6 µmol/kg (median value 13.7 µmol/kg) Sat PC in alveolar
lavages from term newborns (Fig. 1). Sat PC
increased to 1.6% of the term value at 28 days and to 5.7% of the
term value by 29 days. Ventilation did not increase the amount of Sat
PC recovered by alveolar lavage at 27 or 28 days gestation, but the
alveolar Sat PC pool increased to 23% of the term value with
ventilation of 29-day rabbits (P < 0.01 vs. unventilated 29-day rabbits). The median alveolar Sat PC pool
increased 680-fold between 27 days gestation and term.
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The relative amount of SP-C in rabbit alveolar wash samples was
analyzed using antiserum raised against recombinant human SP-C. Figure
2A
shows immunoblot analysis of different amounts of the recombinant SP-C
antigen relative to the anti-SP-C signal from aliquots of a
representative newborn rabbit lavage sample loaded to contain different
amounts of Sat PC. The SP-C from the lavage migrates at the same
position (Mr
5,000) as the recombinant SP-C standard. The pooled lavage samples from
premature and term rabbits (each containing 2.5 µg of Sat PC) were
analyzed using 5 ng of recombinant SP-C as standard. A representative
immunoblot is shown in Fig. 2B. Mature
SP-C is seen to increase relative to Sat PC from 27 days to term. The
amount of SP-C was converted from micrograms to micromoles using a
molecular mass of 3,700 kDa.
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The summaries of the scans for SP-C demonstrated that the amount of
SP-C was very low at 27 and 28 days gestation (Fig.
3). Median values were 1.5 × 106 µmol/kg at 27 days
and 1.2 × 10
5
µmol/kg at 28 days gestation in unventilated rabbits. At 27 days gestation, 3 of 14 alveolar pools and at 28 days gestation, 4 of 20 alveolar pools had no detectable SP-C. The median values did not
increase significantly with ventilation at 27 or 28 days gestation. By 29 days gestation, all rabbits had easily detectable amounts of SP-C, and the median value increased from 1.2 × 10
4 µmol/kg in
unventilated rabbits to 2.4 × 10
3 µmol/kg in ventilated rabbits
(P < 0.01). Median amounts of SP-C increased over 5,000-fold between 27 days gestation and term. The molar
percent of SP-C relative to Sat PC was 0.059 at term (Fig. 3). A
similar value of 0.088 was measured for ventilated rabbits at 28 days.
The molar percent increased from 0.032 at 29 days in unventilated
rabbits to a value of 0.113 with ventilation (P < 0.01).
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At 27 and 28 days gestation, when very little mature SP-C was detectable, proSP-C was evident in the membrane fraction from lung tissue (Fig. 2C). At 29 days and at term, the proSP-C was minimal and the mature SP-C was abundant. In the adult animals, the intensity of the bands for proSP-C and mature SP-C was similar.
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DISCUSSION |
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Surfactant deficiency in the developing lung has been characterized by low amounts of the surfactant phospholipids in alveolar lavages. SP-A is known to also be very low and to remain disproportionately low relative to the phospholipids until term, a finding consistent with low mRNA levels for SP-A until just before term (20). In humans, the mRNA for SP-B appears before SP-A mRNA (13, 14). In rabbits, SP-B and SP-A mRNAs are both detected at day 24 (8). SP-C mRNA and proprotein are anomalous relative to the other surfactant proteins in that they appear at early gestational ages in association with the epithelium where airways branch (14, 25). Subsequently SP-C mRNA (25, 26) and proSP-C (14) are present in distal lung epithelial cells before differentiation into type II cells occurs. The promotor for SP-C has been well characterized and controls this early gestational and spatial expression of the SP-C gene (25). However, the timing of appearance and amounts of mature SP-C in the late-gestation fetal lung have remained unclear, primarily because there have been no satisfactory assays for antibodies with which to quantify mature SP-C.
Based on the pathways for processing the 35-amino acid mature SP-C from its primary approximately 200-amino acid sequence in the mature lung, mature SP-C should not be detectable before type II cells have matured sufficiently to have lamellar bodies (24). The proprotein can be localized immunologically in the trans-Golgi and multivesicular bodies but not in lamellar bodies (22). Mature SP-C is recovered from lamellar bodies and in alveolar lavages in association with lipids (1, 4). Given its extremely hydrophobic behavior, mature SP-C should exist only in association with lipids, and the lamellar bodies provide such an environment within type II cells (24). Therefore, despite steady-state levels of mRNA that are equivalent to those measured at term, the assumption has been that the preterm lung should not be able to process proSP-C to mature SP-C until type II cells have lamellar bodies. Nevertheless, mature SP-C might accumulate in the fetal lung in a presently undescribed location or be present in the fetal air spaces in excess of its normal molar ratio to the surfactant phospholipids.
We measured the amounts of surfactant by quantifying Sat PC. Although the amount of Sat PC relative to total PC or total phospholipids increases as the surfactant system matures, the percent increase in Sat PC is not large and Sat PC remains the best single measure for the size of the surfactant pool (3). We measured SP-C only in the large-aggregate surfactant fraction because SP-C has not been detected in the small- aggregate or vesicular forms of surfactant (17). We also have not been able to detect SP-C with the anti-SP-C antibody used here in the supernatant after removal of large-aggregate surfactant (unpublished observation). SP-C was very low in alveolar washes at 27 and 28 days gestation despite relatively high levels of proSP-C in lung tissue, and short-term ventilation did not release SP-C to the alveolus. By 29 days gestation, alveolar SP-C levels had increased about 70-fold from the 27-day value and proSP-C had decreased in the tissue. Alveolar SP-C increased with ventilation. This pattern indicates that SP-C is not effectively processed until day 29 in the rabbit and suggests a close link between SP-C processing and secretion. The increase in alveolar SP-C paralleled the increase in Sat PC, a result consistent with the concomitant packaging of both surfactant components in lamellar bodies and subsequent cosecretion. The level of proSP-C associated with adult lung membranes is increased compared with the newborn samples (Fig. 2). This may be due to a lower rate of surfactant lipid secretion into the adult airway. The observed increased cellular pool of proSP-C in adult tissue is also consistent with coordinate regulation of SP-C processing and surfactant secretion. Taken together, our results are consistent with what is known about the processing of SP-C and verify that very little mature SP-C is present in the surfactant in the immature fetal lung despite the presence of SP-C mRNA and proSP-C. The preterm alveolar space that is surfactant-lipid deficient also is severely SP-C deficient.
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ACKNOWLEDGEMENTS |
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This work was supported by National Institute of Child Health and Human Development Grant HD-12714.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: A. H. Jobe, Children's Hospital Medical Center, Div. of Pulmonary Biology, 3333 Burnet Ave., Cincinnati, OH 45229-3039 (E-mail: jobea0{at}chmcc.org).
Received 10 February 1999; accepted in final form 12 August 1999.
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