Surfactant Palmitoylmyristoylphosphatidylcholine Is a Marker for Alveolar Size during Disease
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美国呼吸和危急护理医学 2005年第7期
Canadian Institutes of Health Research Group in Lung Development, Lung Biology Program, Hospital for Sick Children Research Institute
Institute of Medical Sciences
Toronto Lung Transplant Program, Toronto General Hospital, University of Toronto, Toronto, Ontario, Canada
Division of Neonatology, Department of Pediatrics, Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland
ABSTRACT
Two common lung-related complications in the neonate are respiratory distress syndrome, which is associated with a failure to generate low surface tension at the aireCliquid interface because of pulmonary surfactant insufficiency, and bronchopulmonary dysplasia (BPD), a chronic lung injury with reduced alveolarization. Surfactant phosphatidylcholine (PC) molecular species composition during alveolarization has not been examined. Mass spectrometry analysis of bronchoalveolar lavage fluid of rodents and humans revealed significant changes in surfactant PC during alveolar development and BPD. In rats, total PC content rose during alveolarization, which was caused by an increase in palmitoylmyristoyl-PC (16:0/14:0PC) concentration. Furthermore, two animal models of BPD exhibited a specific reduction in 16:0/14:0PC content. In humans, 16:0/14:0PC content was specifically decreased in patients with BPD and emphysema compared with patients without alveolar pathology. Palmitoylmyristoyl-PC content increased with increasing intrinsic surfactant curvature, suggesting that it affects surfactant function in the septating lung. The changes in acyl composition of PC were attributed to type II cells producing an altered surfactant during alveolar development. These data are compatible with extracellular surfactant 16:0/14:0PC content being an indicator of alveolar architecture of the lung.
Key Words: alveolar size; bronchopulmonary dysplasia; development; emphysema; surfactant phosphatidylcholine
Pulmonary surfactant is a complex mixture of lipids and proteins that is synthesized and secreted by the alveolar type II epithelial cell into the thin liquid layer that lines the epithelium. Once in the extracellular space, surfactant reduces surface tension at the aireCliquid interface of the lung, a function that requires an appropriate mix of surfactant lipids and the hydrophobic proteins, surfactant proteins B and C (1, 2). Of the surfactant lipids, 80 to 90% are phospholipids, whereas the rest are neutral lipids. The most abundant phospholipid species is phosphatidylcholine (PC), with dipalmitoyl-PC (16:0/16:0PC) being important in attaining surface tensions near 0 N/m (3eC7). Although saturated palmitoylmyristoyl-PC (16:0/14:0PC) and monounsaturated palmitoylpalmitoleoyl-PC (16:0/16:1PC) are also prevalent in most mammalian lung surfactants (7), their contribution to surfactant function is not understood. In vitro studies have demonstrated that 16:0/16:0PC does not spread well at the aireCliquid interface (8, 9). One possibility is that 16:0/14:0PC and 16:0/16:1PC assist in the surface spreading of 16:0/16:0PC (10, 11). Recent data have suggested that 16:0/14:0PC and 16:0/16:1PC may contribute to dynamic surfactant functions during mammalian respiration (4). The fractional concentrations of both PC species in lung surfactant have been found to correlate with respiratory rates in mammals (4). With increasing respiratory rates, both 16:0/14:0PC and 16:0/16:1PC concentrations in surfactant are increased. Thus, the fractional concentrations of 16:0/14:0PC and 16:0/16:1PC in surfactant are adapted to the physiologic needs of the mammalian lung (4).
There are two events during lung development that may have specific surfactant needs. First, at birth, the lungs require large amounts of surfactant to convert the fluid-filled saccules into gas-exchange units with a stable aireCliquid interface. Failure to establish a low surface-tension aireCliquid interface at the distal airspace results in the respiratory distress syndrome, a common complication of premature birth as a result of delayed lung fluid clearance and/or pulmonary surfactant insufficiency (12, 13). The absolute and fractional changes in concentrations of functionally important PC species (16:0/16:0PC, 16:0/14:0PC, and 16:0/16:1PC) immediately after birth have not been reported. Second, in several mammalian species, distal lung development proceeds postnatally, such that the alveoli are formed exclusively after birth (e.g., rats and mice) or predominantly after birth (e.g., humans). The process of alveolarization involves the division of the preexisting voluminous terminal air sacs (saccules) into smaller units, the alveoli, by secondary septa. These septa grow out from the saccular walls into the airspaces in a centripetal manner. As a result, there is an increase in the number of terminal gas-exchange units (14). Although, these newly formed alveoli have less volume, there is a substantial net increase in total surface area (14). In rats (14) and mice (15), the bulk of secondary septation takes place between Postnatal Days 4 and 14. Human alveolarization occurs mainly between 36 weeks of gestation and 18 months of age (16). The completion of alveolarization results in an increased number of terminal airway units, which continue to grow in size to further expand the surface area into adult life. Whether these morphologic surface changes during alveolarization impact on the composition of functionally important surfactant PC species is also unknown. Therefore, we performed a thorough developmental analysis of PC species in surfactants of rat lung from Fetal Day 19 to maturity (Postnatal Day 22). In addition, we measured surfactant PC species in neonatal rat models of reduced alveolarization and in infants with bronchopulmonary dysplasia (BPD).
METHODS
Animal Models
Timed-pregnant female Wistar rats and C57/Bl6 mice were obtained from Charles River (St. Constant, PQ, Canada). All animal protocols were in accordance with Canadian Counsel of Animal Care guidelines and were approved by the Animal Care and Use Committee of the Hospital for Sick Children.
Developmental profile.
At Fetal Days 19 and 22 (term, 23 days), the pregnant rat was anesthetized and the pups delivered by cesarean section, while postpartum rodents were removed from their mother directly before being killed.
Dexamethasone treatment.
Newborn Wistar rats were injected daily for 4 days with dexamethasone (Sabex, Boucherville, PQ, Canada) diluted in isotonic saline starting at Postnatal Day 1. The dosage was reduced by half every day, with the starting dose of 0.1 mg dexamethasone/kg bodyweight (17).
Hyperoxia exposure.
The oxygen treatment was performed by housing the rat pups with their mother in chambers containing either 21 or 60% oxygen, starting at Day 1 until day of lavage (i.e., Postnatal Day 4, 7, 10, or 14) (18).
Bronchoalveolar Lavage Fluid from Rodents
After killing the animals, a needle (blunted tip) was inserted through a tracheostomy and the lungs were lavaged with a buffer composed of phosphate-buffered saline augmented with 0.05 mg/ml of 70 kD dextraneCfluorescein isothiocyanate (Molecular Probes, Burlington, ON, Canada). The inert fluorescent marker was included to determine lavage recovery (see Figure E1 on the online supplement).
Tracheal Aspirate and Bronchoalveolar Lavage Fluid from Humans
Tracheal aspirates from infants were obtained by irrigation through the endotracheal tube using 1 ml of saline. Respiratory distress syndrome was defined as a requirement for exogenous surfactant at the time of birth for babies 27 weeks' gestational age or older. For babies younger than 27 weeks' gestational age, respiratory distress syndrome was defined as the ongoing need for mechanical ventilation after exogenous surfactant therapy. Although the diagnosis was made at the time of birth, the samples were collected from 29 to 42 weeks' corrected gestational age (median, 31 weeks; n = 19). BPD was defined by a consistent clinical course and x-ray changes. This was later confirmed by a continued requirement for supplemental oxygen at 36 weeks' corrected gestational age. The samples of patients with BPD were collected between 28 and 43 weeks of gestation (median, 32 weeks; n = 8). Prenatal steroids (Celestone; Schering, Berlin, Germany) were administered to 21 of 28 patients, equally distributed between the respiratory distress syndrome and BPD groups. For adult patients, bronchoscopy was done using 50 ml of saline for bronchoalveolar lavage (BAL). Samples from patients with emphysema (n = 3) and patients with idiopathic pulmonary fibrosis (n = 3) were collected before lung transplantation. Control samples were taken before transplantation from patients with no alveolar complications (n = 8) and from patients with lung tumors (n = 4) with little or no chronic obstructive pulmonary disease. Samples were spun at 1,000 g for 5 minutes to remove cellular material. All patient samples were obtained in accordance with Health Canada's Research Ethics Board guidelines.
Mass Spectral Analysis of PC
BAL fluid (BALF) samples were spiked with 1 e of deuterated 16:0/16:0PC (Avanti Polar Lipids, Alabaster, AL) as an internal standard, and then extracted (19). Lipids were analyzed using an API4000 mass spectrometer (MDS Sciex, Concord, ON, Canada) (20).
Light Scattering
BALF samples, containing 50 nmol/L of PC, were extracted, and lipids were dried under nitrogen. Lipid samples were then reconstituted in 1 ml of saline at 37°C followed by bath sonication. After one freezeeCthaw cycle, the vesicle size was determined by dynamic light scattering (21) using a Malvern Mastersizer X (Malvern Instruments Ltd., Worcestershire, UK).
Laser Capture Microdissection
Cryo-embedded lung sections were processed as previously described (20). Type II cells were visualized using a rabbit polyclonal antibody against pro-N-SP-C (provided by Dr. Michael Beers, University of Pennsylvania, Philadelphia, PA) followed by a fluorescein isothiocyanateeCconjugated secondary goat antirabbit IgG antibody (Calbiochem, San Diego, CA). Approximately 200 type II cells were captured using a PixCell II system (Arcturus, Mountain View, CA); lipids were extracted and analyzed by mass spectrometry.
Statistics
All values are shown as mean ± SE. Statistical analysis was done by Student's t test or, for comparison of more than two groups, by one-way analysis of variance followed by Duncan's multiple range comparison test, with significance defined as p less than 0.05.
RESULTS AND DISCUSSION
Developmental Profile of Total PC
BAL was performed on rats at differing gestational and postpartum ages. Total PC concentration was determined by the sum of the concentrations of all individual PC species. As can be seen in Figure 1a, PC content in BALF varied tremendously during fetal and postnatal lung development. In agreement with previous reports (see Reference 22), the amount of extracellular surfactant PC increased significantly (40-fold) between 19 and 22 days' gestation (Figure 1b). A further 10-fold increase in total PC content of BALF occurred within the first 2 hours after birth (Figure 1c). This immediate rise in surfactant PC after birth may be attributed to two factors. First, the decrease in alveolar fluid via clearance would concentrate the components of the bronchoalveolar compartment, including surfactant. At or shortly before birth, the lung switches from a fluid-secreting to a fluid-absorbing organ. Although much of the lung fluid is cleared within 2 hours of birth, there is evidence that the process of lung fluid adsorption is a more protracted process, lasting more than 40 hours in the rat (23). Second, it is well known that mechanical forces increase surfactant secretion (24eC26). Lung expansion caused by the first deep sigh or onset of breathing has been shown to stimulate the release of preformed lamellar bodies into the extracellular space (27, 28).
Interestingly, the concentration of PC in BALF decreased slightly after 2 hours postpartum, but then increased and reached maximal levels at 24 hours after birth after which it declined and reached mature levels at Day 4 postpartum (Figure 1c). We hypothesize that this second postnatal surge in PC content in BALF is caused by the secretion of newly synthesized surfactant PC. Earlier observations that choline incorporation into rat lung tissue PC peaked on the first day after birth support this idea (see Reference 22). The synthesis of new lamellar bodies has been estimated to require approximately 6 hours (29, 30), which would explain the plateau between the two peaks at 2 and 24 hours postpartum. Thus, the requirement for surfactant during early extrauterine life is met by release of preformed lamellar bodies within the first few hours of breathing followed by massive synthesis and secretion by type II cells of new surfactant material. Alternatively, the second peak could be a secretion from more slowly released surfactant pool (31). How type II cells sense this need for new surfactant within 2 hours after the onset of breathing remains an enigma.
Another increase of total PC content in BALF occurred after Postnatal Day 4, with a peak at Day 12 and a subsequent decline until Day 19 when adult levels were reached (Figure 1a). In rats, the bulk of alveolarization takes place between Days 4 and 14 (14). To our knowledge, this concomitant relationship between PC concentration and alveolarization has never been reported. The enlargement in surface area that occurs during alveolarization would require an increasing amount of surfactant. The increase in the amount of surfactant PC during alveolarization suggests that there may be a coregulation of surfactant and septal formation. Type II cell numbers are believed to peak during alveolarization (32). The increased number of type II cells could account for increased surfactant production during this time period. However, the increase in PC concentration during alveolarization is primarily caused by 16:0/14:0PC and not 16:0/16:0PC (Figure 2d).
Developmental Profile of Major PC Species
The three predominant species of PC in surfactant (16:0/16:0PC, 16:0/16:1PC, and 16:0/14:0PC) were examined by absolute concentration and percentage distribution (Figure 2). Before birth, the proportion of 16:0/16:0PC increased from 23% at Day 19 to 40% at birth (Figure 2d, Table 1), whereas the proportion of larger acyl chain unsaturated PC (16:0/18:1PC, 18:0/18:2PC, 16:0/20:4PC, 18:0/22:6PC, and 18:1/18:2PC) declined (Table 1). After birth, the fractional concentration of 16:0/16:0PC remained consistently close to 40% (Figure 2d, Table 1), although absolute values altered substantially (Figure 2a). These data indicate that, for the rat, 16:0/16:0PC ratios with respect to total PC content is regulated to a near constant value (Figure 2a). In many mammals, 16:0/16:0PC content in surfactant fluctuates between 35 and 60% (rabbits, 35.6% [6]; humans, 54% [6, 33]), suggesting that it is a crucial concentration for optimal surface-active function in vivo.
A recent report has shown that surfactant from piglets is enriched in 16:0/16:1PC and 16:0/14:0PC relative to adult pigs (34). Herein, we demonstrate for the first time that these two PC species have a distinct profile during fetal and postnatal development (Figures 2beC2d). The fractional concentration of 16:0/16:1PC in BALF was greatest at birth (33%) and diminished postpartum (Figures 2b and 2d, Table 1). There was no major change in its concentration between Postnatal Days 7 and 22 (Figure 2d). The high concentration of 16:0/16:1 at birth (Figure 2b) may aid in the establishment of the first aireCliquid interface that is required at this time. This establishment requires a rapid adsorption of surfactant to the interface. Besides Surfactant proteins B and C (35), unsaturated PC, such as 16:0/18:1PC, has also been shown to improve 16:0/16:0PC adsorption at the aireCliquid interface (11). Although 16:0/18:1PC may improve the adsorption rate of surfactant to the interface, we found that its fractional concentration varied little throughout development (data not shown). Moreover, its concentration was far below that of 16:0/16:1PC around birth (33% for 16:0/16:1PC vs. 9% for 16:0/18:1PC). Considering that 16:0/16:1PC, like 16:0/18:1, has a greater molecule-to-water ratio at the watereCair interphase than 16:0/16:0PC at a given pressure, it is likely to have better adsorption characteristics than 16:0/16:0PC (36eC38). Therefore, we speculate that 16:0/16:1PC plays an important role in forming the pulmonary surfactant film immediately after birth.
The 16:0/14:0PC amount in BALF increased at birth consistently with the rise in concentrations of 16:0/16:0PC and 16:0/16:1PC (Figure 2c, Table 1). However, 16:0/14:0PC content in BALF peaked between Days 12 and 14 postpartum (Figure 2c). Fractional 16:0/14:0PC concentrations were increased from Postnatal Days 7 to 14 (Figure 2d), which corresponds to the alveolarization period in the rat (14). In fact, the general rise in total PC content during this time (Figure 1a) was primarily accounted for by the increase in 16:0/14:0PC (Figure 2d). The function of 16:0/14:0PC in pulmonary surfactant is unclear. It has a similar molecule-to-water ratio at the watereCair interphase as 16:0/16:0PC at a given pressure (36, 37). The marginal chain asymmetry will likely not result in a pronounced difference over 16:0/16:0PC with respect to adsorption properties. Thus, it is unlikely that 16:0/14:0PC, in contrast to 16:0/16:1PC, enhances the aireCliquid adsorption rates of 16:0/16:0PC.
Considering the unclear role for 16:0/14:0PC in surfactant function, we investigated whether the rise of 16:0/14:0PC during alveolarization was specific to the rat. Therefore, we analyzed BALF from mice at late gestation (Day 18) and around the peak (Postnatal Day 10) of alveolarization (15). Mouse and rat BALF had similar fractional 16:0/16:0PC levels at fetal, postnatal (alveolar period), and mature time points (Figure 3a). Likewise, there was a similar trend between the two rodents for high 16:0/16:1PC content in fetal samples and high 16:0/14:0PC content during alveolarization (Figures 3b and 3c). This consistency between the two rodent species suggests that the relatively high 16:0/16:1PC content of surfactant at birth as well as the relatively high 16:0/14:0PC content of surfactant during alveolarization is a general phenomenon, at least in rodents.
Surfactant 16:0/14:0PC in Rat Models of Disturbed Alveolarization
As stated above, surfactant 16:0/14:0PC increased during the period of alveolarization in the rat. To determine the nature of this relationship, we examined BALF from two rat models of diminished alveolarization. The two models we used were postnatal exposure to either dexamethasone or 60% oxygen. Postnatal administration of dexamethasone to rats has been described to result in a reduced alveolarization (17, 39eC41). We choose to use a high-dose short-term treatment strategy, which results, at Day 10, in a significant decreased parenchymal complexity with larger and fewer alveoli compared with control subjects (17). Several studies have shown that neonatal hyperoxia of mice and rats also results in diminished alveolarization (18, 42eC44). Exposure of neonatal rats to 60% oxygen resulted in a significant reduction of total PC in BALF at Postnatal Day 10 (Figure 4a). By Postnatal Day 14, total PC values were no longer significantly different between 60% oxygen- and air-exposed animals (Figure 4e). Similar observations of reduced surfactant PC have been reported for neonatal rabbits exposed to 98% oxygen (45), although this effect could be the result of acute cellular injury. The content of individual PC molecules, including 16:0/16:0PC, 16:0/14:0PC, and 16:0/16:1PC, were also reduced in BALF of rats exposed to 60% oxygen (Figures 4beC4h). The overall reduction in surfactant PC synthesis may be caused by several factors. Hyperoxia is known to induce the release of a number of cytokines (46). Some of these cytokines—in particular, tumor necrosis factor —have been shown to reduce the activity of the rate-limiting enzyme in PC synthesis, CTP:phosphocholine cytidylyltransferase (47eC50). In addition, oxidant stress has been shown to affect another crucial lipid-synthesizing enzyme, glycerol phosphate acyltransferase (51). Alternatively, hyperoxia increases lipid peroxidation in rat type II cells (52), and therefore, type II cells may be shunt their lipid production from surfactant synthesis to cellular membrane repair. In contrast to hyperoxia, postpartum dexamethasone treatment resulted in a significant increase of total PC content in BALF at Day 10 (Figure 4a). In particular, monounsaturated PC molecules, such as 16:0/16:1PC (Figure 4c) and 16:0/18:1 (data not shown), were elevated in BALF of dexamethasone-treated neonatal rats. Similar observations have been reported for liver PC of dexamethasone-treated rats (53). Saturated PC species, including 16:0/16:0PC, were also elevated in BALF of dexamethasone-treated neonatal rats (Figure 4b), with the exception of 16:0/14:0PC, which was significantly reduced (Figure 4d). Interestingly, 16:0/14:0PC (Figure 4h) was the only PC species (Figures 4eeC4g) significantly reduced after 14 days of 60% oxygen exposure. Thus, the two rat models of reduced alveolarization had contrasting effects on surfactant PC production (i.e., upregulation with dexamethasone and downregulation with 60% oxygen). The only consistency with respect to surfactant PC was a constant decrease in 16:0/14:0PC concentration. Taken together, the results suggest that surfactant 16:0/14:0PC concentrations relate to the alveolarization process not only during normal lung development but also in two different models of diminished alveolar formation.
Surfactant 16:0/14:0PC in Human Diseases of Disturbed Alveolar Architecture
To evaluate the three predominant species of PC in human surfactant, we gathered tracheal aspirates from intubated neonates and BALF from adults. Data were subdivided on the basis of pathology. Tracheal aspirates were obtained from infants with either respiratory distress syndrome or BPD. BALF was obtained from adult patients with emphysema or idiopathic pulmonary fibrosis, and from patients after lung transplantation with no pronounced alveolar complication. These samples were compared with BALF obtained from patients with lung cancer who had normal alveolar architecture. Samples of patients with emphysema and infants with BPD displayed significantly reduced surfactant 16:0/14:0PC levels compared with all other patient groups tested (Figure 5b). Dipalmitoyl-PC did not significantly differ between the patient groups (Figure 5a). Thus, the changes in 16:0/14:0PC content in the emphysemic and BPD groups are likely not the result of a general loss of surfactant. Emphysema is characterized by abnormal, permanent enlargement of airspaces distal from the terminal bronchi, whereas one of the hallmarks of BPD seen in premature infants is alveolar simplification (i.e., larger but fewer alveoli). Although the human samples, specifically of adults, are limited, the reduced 16:0/14:0PC content in lavage fluid of BPD and emphysema is in line with the rat models of reduced alveolarization. As such, the results suggest that surfactant 16:0/14:0PC content in humans also relates to distal airspace architecture of the lung.
Possible Role for 16:0/14:0 PC in Surfactant during Alveolarization
The main feature of alveolarization is the subdivision of the preexisting voluminous saccules by septation, which leads to smaller units (alveoli) and an increased total surface area. These units then have the potential to increase in size during growth of the animal. One of the implications of saccular subdivision is an increase in alveolar curvature. Two groups have reported alterations in alveolar diameter (14) and volume (54) during the period of alveolarization in the rat (14, 54, 55). When we plotted radii calculated from the reported alveolar dimensions during rat development against 16:0/14:0PC in rat BALF, a striking relationship between 16:0/14:0PC content and the curvature was found (Figure 6a).
To further investigate this relationship, we performed light-scattering analysis on liposomes formed from lipids extracted of BALF. Lipids in BALF of Day 10 neonatal rats, which have a high surfactant 16:0/14:0PC content (Figure 4d), formed liposomes with an average particle size of 9.0 ± 0.4 e. In contrast, lipids in BALF from dexamethasone- and 60% oxygeneCtreated rats, which have a lower surfactant 16:0/14:0PC content (Figure 4d), formed significantly larger liposomes (13.2 ± 0.7 and 12.6 ± 0.6 e, respectively). The BALF liposomes of 22-day-old rats (14 ± 0.7 e) were significantly larger than those of 10-day-old rats, consistent with the lower 16:0/14:0PC content (Figures 2c and 2d). When we plotted the radii of BALF liposomes against BALF 16:0/14:0PC content, a strong correlation (r2 = 0.998) was found (Figure 6b), suggesting that 16:0/14:0PC increases the curving capacity of surfactant lipids. This would explain a potential basis for the increased 16:0/14:0PC levels in surfactant of higher curved airspaces that occurs during alveolarization. However, some caution is warranted because we did not consider changes in other variables, including surfactant proteins B and C.
Intrinsic curvature in membranes can be influenced by small polar head lipids (56) or by asymmetric acyl chain length (57eC59). Freeze fracture analysis of PC has shown that 16:0/16:0PC liposomes have a threefold greater radius compared with 16:0/14:0PC liposomes (60). The acyl chains of 16:0/14:0PC will not have the same surface packing as 16:0/16:0PC at 37°C, but because of its distinct acyl packing characteristics, it may obtain high surface pressures when spread on a highly curved interface. Therefore, we hypothesize that 16:0/14:0PC improves surfactant function during secondary septation, which is associated with more curved surface areas.
Finally, we investigated whether type II cells control acyl composition of PC during alveolarization. Using laser-capture microscopy and mass spectral lipid analysis, we found that 16:0/14:0PC content of rat type II cells increased postnatally from Day 7, peaked at Days 12 through 14, and subsequently declined until Day 21 when adult levels were reached (Figure 6c). The similarity between cellular and BALF 16:0/14:0PC content during the alveolarization period suggests that the lipid changes in the BALF are caused by type II cells producing a different surfactant. How the type II cells sense architectural changes and produce acyl-specific PC during alveolarization remains to be elucidated.
This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org
These authors contributed equally to this study.
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Institute of Medical Sciences
Toronto Lung Transplant Program, Toronto General Hospital, University of Toronto, Toronto, Ontario, Canada
Division of Neonatology, Department of Pediatrics, Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland
ABSTRACT
Two common lung-related complications in the neonate are respiratory distress syndrome, which is associated with a failure to generate low surface tension at the aireCliquid interface because of pulmonary surfactant insufficiency, and bronchopulmonary dysplasia (BPD), a chronic lung injury with reduced alveolarization. Surfactant phosphatidylcholine (PC) molecular species composition during alveolarization has not been examined. Mass spectrometry analysis of bronchoalveolar lavage fluid of rodents and humans revealed significant changes in surfactant PC during alveolar development and BPD. In rats, total PC content rose during alveolarization, which was caused by an increase in palmitoylmyristoyl-PC (16:0/14:0PC) concentration. Furthermore, two animal models of BPD exhibited a specific reduction in 16:0/14:0PC content. In humans, 16:0/14:0PC content was specifically decreased in patients with BPD and emphysema compared with patients without alveolar pathology. Palmitoylmyristoyl-PC content increased with increasing intrinsic surfactant curvature, suggesting that it affects surfactant function in the septating lung. The changes in acyl composition of PC were attributed to type II cells producing an altered surfactant during alveolar development. These data are compatible with extracellular surfactant 16:0/14:0PC content being an indicator of alveolar architecture of the lung.
Key Words: alveolar size; bronchopulmonary dysplasia; development; emphysema; surfactant phosphatidylcholine
Pulmonary surfactant is a complex mixture of lipids and proteins that is synthesized and secreted by the alveolar type II epithelial cell into the thin liquid layer that lines the epithelium. Once in the extracellular space, surfactant reduces surface tension at the aireCliquid interface of the lung, a function that requires an appropriate mix of surfactant lipids and the hydrophobic proteins, surfactant proteins B and C (1, 2). Of the surfactant lipids, 80 to 90% are phospholipids, whereas the rest are neutral lipids. The most abundant phospholipid species is phosphatidylcholine (PC), with dipalmitoyl-PC (16:0/16:0PC) being important in attaining surface tensions near 0 N/m (3eC7). Although saturated palmitoylmyristoyl-PC (16:0/14:0PC) and monounsaturated palmitoylpalmitoleoyl-PC (16:0/16:1PC) are also prevalent in most mammalian lung surfactants (7), their contribution to surfactant function is not understood. In vitro studies have demonstrated that 16:0/16:0PC does not spread well at the aireCliquid interface (8, 9). One possibility is that 16:0/14:0PC and 16:0/16:1PC assist in the surface spreading of 16:0/16:0PC (10, 11). Recent data have suggested that 16:0/14:0PC and 16:0/16:1PC may contribute to dynamic surfactant functions during mammalian respiration (4). The fractional concentrations of both PC species in lung surfactant have been found to correlate with respiratory rates in mammals (4). With increasing respiratory rates, both 16:0/14:0PC and 16:0/16:1PC concentrations in surfactant are increased. Thus, the fractional concentrations of 16:0/14:0PC and 16:0/16:1PC in surfactant are adapted to the physiologic needs of the mammalian lung (4).
There are two events during lung development that may have specific surfactant needs. First, at birth, the lungs require large amounts of surfactant to convert the fluid-filled saccules into gas-exchange units with a stable aireCliquid interface. Failure to establish a low surface-tension aireCliquid interface at the distal airspace results in the respiratory distress syndrome, a common complication of premature birth as a result of delayed lung fluid clearance and/or pulmonary surfactant insufficiency (12, 13). The absolute and fractional changes in concentrations of functionally important PC species (16:0/16:0PC, 16:0/14:0PC, and 16:0/16:1PC) immediately after birth have not been reported. Second, in several mammalian species, distal lung development proceeds postnatally, such that the alveoli are formed exclusively after birth (e.g., rats and mice) or predominantly after birth (e.g., humans). The process of alveolarization involves the division of the preexisting voluminous terminal air sacs (saccules) into smaller units, the alveoli, by secondary septa. These septa grow out from the saccular walls into the airspaces in a centripetal manner. As a result, there is an increase in the number of terminal gas-exchange units (14). Although, these newly formed alveoli have less volume, there is a substantial net increase in total surface area (14). In rats (14) and mice (15), the bulk of secondary septation takes place between Postnatal Days 4 and 14. Human alveolarization occurs mainly between 36 weeks of gestation and 18 months of age (16). The completion of alveolarization results in an increased number of terminal airway units, which continue to grow in size to further expand the surface area into adult life. Whether these morphologic surface changes during alveolarization impact on the composition of functionally important surfactant PC species is also unknown. Therefore, we performed a thorough developmental analysis of PC species in surfactants of rat lung from Fetal Day 19 to maturity (Postnatal Day 22). In addition, we measured surfactant PC species in neonatal rat models of reduced alveolarization and in infants with bronchopulmonary dysplasia (BPD).
METHODS
Animal Models
Timed-pregnant female Wistar rats and C57/Bl6 mice were obtained from Charles River (St. Constant, PQ, Canada). All animal protocols were in accordance with Canadian Counsel of Animal Care guidelines and were approved by the Animal Care and Use Committee of the Hospital for Sick Children.
Developmental profile.
At Fetal Days 19 and 22 (term, 23 days), the pregnant rat was anesthetized and the pups delivered by cesarean section, while postpartum rodents were removed from their mother directly before being killed.
Dexamethasone treatment.
Newborn Wistar rats were injected daily for 4 days with dexamethasone (Sabex, Boucherville, PQ, Canada) diluted in isotonic saline starting at Postnatal Day 1. The dosage was reduced by half every day, with the starting dose of 0.1 mg dexamethasone/kg bodyweight (17).
Hyperoxia exposure.
The oxygen treatment was performed by housing the rat pups with their mother in chambers containing either 21 or 60% oxygen, starting at Day 1 until day of lavage (i.e., Postnatal Day 4, 7, 10, or 14) (18).
Bronchoalveolar Lavage Fluid from Rodents
After killing the animals, a needle (blunted tip) was inserted through a tracheostomy and the lungs were lavaged with a buffer composed of phosphate-buffered saline augmented with 0.05 mg/ml of 70 kD dextraneCfluorescein isothiocyanate (Molecular Probes, Burlington, ON, Canada). The inert fluorescent marker was included to determine lavage recovery (see Figure E1 on the online supplement).
Tracheal Aspirate and Bronchoalveolar Lavage Fluid from Humans
Tracheal aspirates from infants were obtained by irrigation through the endotracheal tube using 1 ml of saline. Respiratory distress syndrome was defined as a requirement for exogenous surfactant at the time of birth for babies 27 weeks' gestational age or older. For babies younger than 27 weeks' gestational age, respiratory distress syndrome was defined as the ongoing need for mechanical ventilation after exogenous surfactant therapy. Although the diagnosis was made at the time of birth, the samples were collected from 29 to 42 weeks' corrected gestational age (median, 31 weeks; n = 19). BPD was defined by a consistent clinical course and x-ray changes. This was later confirmed by a continued requirement for supplemental oxygen at 36 weeks' corrected gestational age. The samples of patients with BPD were collected between 28 and 43 weeks of gestation (median, 32 weeks; n = 8). Prenatal steroids (Celestone; Schering, Berlin, Germany) were administered to 21 of 28 patients, equally distributed between the respiratory distress syndrome and BPD groups. For adult patients, bronchoscopy was done using 50 ml of saline for bronchoalveolar lavage (BAL). Samples from patients with emphysema (n = 3) and patients with idiopathic pulmonary fibrosis (n = 3) were collected before lung transplantation. Control samples were taken before transplantation from patients with no alveolar complications (n = 8) and from patients with lung tumors (n = 4) with little or no chronic obstructive pulmonary disease. Samples were spun at 1,000 g for 5 minutes to remove cellular material. All patient samples were obtained in accordance with Health Canada's Research Ethics Board guidelines.
Mass Spectral Analysis of PC
BAL fluid (BALF) samples were spiked with 1 e of deuterated 16:0/16:0PC (Avanti Polar Lipids, Alabaster, AL) as an internal standard, and then extracted (19). Lipids were analyzed using an API4000 mass spectrometer (MDS Sciex, Concord, ON, Canada) (20).
Light Scattering
BALF samples, containing 50 nmol/L of PC, were extracted, and lipids were dried under nitrogen. Lipid samples were then reconstituted in 1 ml of saline at 37°C followed by bath sonication. After one freezeeCthaw cycle, the vesicle size was determined by dynamic light scattering (21) using a Malvern Mastersizer X (Malvern Instruments Ltd., Worcestershire, UK).
Laser Capture Microdissection
Cryo-embedded lung sections were processed as previously described (20). Type II cells were visualized using a rabbit polyclonal antibody against pro-N-SP-C (provided by Dr. Michael Beers, University of Pennsylvania, Philadelphia, PA) followed by a fluorescein isothiocyanateeCconjugated secondary goat antirabbit IgG antibody (Calbiochem, San Diego, CA). Approximately 200 type II cells were captured using a PixCell II system (Arcturus, Mountain View, CA); lipids were extracted and analyzed by mass spectrometry.
Statistics
All values are shown as mean ± SE. Statistical analysis was done by Student's t test or, for comparison of more than two groups, by one-way analysis of variance followed by Duncan's multiple range comparison test, with significance defined as p less than 0.05.
RESULTS AND DISCUSSION
Developmental Profile of Total PC
BAL was performed on rats at differing gestational and postpartum ages. Total PC concentration was determined by the sum of the concentrations of all individual PC species. As can be seen in Figure 1a, PC content in BALF varied tremendously during fetal and postnatal lung development. In agreement with previous reports (see Reference 22), the amount of extracellular surfactant PC increased significantly (40-fold) between 19 and 22 days' gestation (Figure 1b). A further 10-fold increase in total PC content of BALF occurred within the first 2 hours after birth (Figure 1c). This immediate rise in surfactant PC after birth may be attributed to two factors. First, the decrease in alveolar fluid via clearance would concentrate the components of the bronchoalveolar compartment, including surfactant. At or shortly before birth, the lung switches from a fluid-secreting to a fluid-absorbing organ. Although much of the lung fluid is cleared within 2 hours of birth, there is evidence that the process of lung fluid adsorption is a more protracted process, lasting more than 40 hours in the rat (23). Second, it is well known that mechanical forces increase surfactant secretion (24eC26). Lung expansion caused by the first deep sigh or onset of breathing has been shown to stimulate the release of preformed lamellar bodies into the extracellular space (27, 28).
Interestingly, the concentration of PC in BALF decreased slightly after 2 hours postpartum, but then increased and reached maximal levels at 24 hours after birth after which it declined and reached mature levels at Day 4 postpartum (Figure 1c). We hypothesize that this second postnatal surge in PC content in BALF is caused by the secretion of newly synthesized surfactant PC. Earlier observations that choline incorporation into rat lung tissue PC peaked on the first day after birth support this idea (see Reference 22). The synthesis of new lamellar bodies has been estimated to require approximately 6 hours (29, 30), which would explain the plateau between the two peaks at 2 and 24 hours postpartum. Thus, the requirement for surfactant during early extrauterine life is met by release of preformed lamellar bodies within the first few hours of breathing followed by massive synthesis and secretion by type II cells of new surfactant material. Alternatively, the second peak could be a secretion from more slowly released surfactant pool (31). How type II cells sense this need for new surfactant within 2 hours after the onset of breathing remains an enigma.
Another increase of total PC content in BALF occurred after Postnatal Day 4, with a peak at Day 12 and a subsequent decline until Day 19 when adult levels were reached (Figure 1a). In rats, the bulk of alveolarization takes place between Days 4 and 14 (14). To our knowledge, this concomitant relationship between PC concentration and alveolarization has never been reported. The enlargement in surface area that occurs during alveolarization would require an increasing amount of surfactant. The increase in the amount of surfactant PC during alveolarization suggests that there may be a coregulation of surfactant and septal formation. Type II cell numbers are believed to peak during alveolarization (32). The increased number of type II cells could account for increased surfactant production during this time period. However, the increase in PC concentration during alveolarization is primarily caused by 16:0/14:0PC and not 16:0/16:0PC (Figure 2d).
Developmental Profile of Major PC Species
The three predominant species of PC in surfactant (16:0/16:0PC, 16:0/16:1PC, and 16:0/14:0PC) were examined by absolute concentration and percentage distribution (Figure 2). Before birth, the proportion of 16:0/16:0PC increased from 23% at Day 19 to 40% at birth (Figure 2d, Table 1), whereas the proportion of larger acyl chain unsaturated PC (16:0/18:1PC, 18:0/18:2PC, 16:0/20:4PC, 18:0/22:6PC, and 18:1/18:2PC) declined (Table 1). After birth, the fractional concentration of 16:0/16:0PC remained consistently close to 40% (Figure 2d, Table 1), although absolute values altered substantially (Figure 2a). These data indicate that, for the rat, 16:0/16:0PC ratios with respect to total PC content is regulated to a near constant value (Figure 2a). In many mammals, 16:0/16:0PC content in surfactant fluctuates between 35 and 60% (rabbits, 35.6% [6]; humans, 54% [6, 33]), suggesting that it is a crucial concentration for optimal surface-active function in vivo.
A recent report has shown that surfactant from piglets is enriched in 16:0/16:1PC and 16:0/14:0PC relative to adult pigs (34). Herein, we demonstrate for the first time that these two PC species have a distinct profile during fetal and postnatal development (Figures 2beC2d). The fractional concentration of 16:0/16:1PC in BALF was greatest at birth (33%) and diminished postpartum (Figures 2b and 2d, Table 1). There was no major change in its concentration between Postnatal Days 7 and 22 (Figure 2d). The high concentration of 16:0/16:1 at birth (Figure 2b) may aid in the establishment of the first aireCliquid interface that is required at this time. This establishment requires a rapid adsorption of surfactant to the interface. Besides Surfactant proteins B and C (35), unsaturated PC, such as 16:0/18:1PC, has also been shown to improve 16:0/16:0PC adsorption at the aireCliquid interface (11). Although 16:0/18:1PC may improve the adsorption rate of surfactant to the interface, we found that its fractional concentration varied little throughout development (data not shown). Moreover, its concentration was far below that of 16:0/16:1PC around birth (33% for 16:0/16:1PC vs. 9% for 16:0/18:1PC). Considering that 16:0/16:1PC, like 16:0/18:1, has a greater molecule-to-water ratio at the watereCair interphase than 16:0/16:0PC at a given pressure, it is likely to have better adsorption characteristics than 16:0/16:0PC (36eC38). Therefore, we speculate that 16:0/16:1PC plays an important role in forming the pulmonary surfactant film immediately after birth.
The 16:0/14:0PC amount in BALF increased at birth consistently with the rise in concentrations of 16:0/16:0PC and 16:0/16:1PC (Figure 2c, Table 1). However, 16:0/14:0PC content in BALF peaked between Days 12 and 14 postpartum (Figure 2c). Fractional 16:0/14:0PC concentrations were increased from Postnatal Days 7 to 14 (Figure 2d), which corresponds to the alveolarization period in the rat (14). In fact, the general rise in total PC content during this time (Figure 1a) was primarily accounted for by the increase in 16:0/14:0PC (Figure 2d). The function of 16:0/14:0PC in pulmonary surfactant is unclear. It has a similar molecule-to-water ratio at the watereCair interphase as 16:0/16:0PC at a given pressure (36, 37). The marginal chain asymmetry will likely not result in a pronounced difference over 16:0/16:0PC with respect to adsorption properties. Thus, it is unlikely that 16:0/14:0PC, in contrast to 16:0/16:1PC, enhances the aireCliquid adsorption rates of 16:0/16:0PC.
Considering the unclear role for 16:0/14:0PC in surfactant function, we investigated whether the rise of 16:0/14:0PC during alveolarization was specific to the rat. Therefore, we analyzed BALF from mice at late gestation (Day 18) and around the peak (Postnatal Day 10) of alveolarization (15). Mouse and rat BALF had similar fractional 16:0/16:0PC levels at fetal, postnatal (alveolar period), and mature time points (Figure 3a). Likewise, there was a similar trend between the two rodents for high 16:0/16:1PC content in fetal samples and high 16:0/14:0PC content during alveolarization (Figures 3b and 3c). This consistency between the two rodent species suggests that the relatively high 16:0/16:1PC content of surfactant at birth as well as the relatively high 16:0/14:0PC content of surfactant during alveolarization is a general phenomenon, at least in rodents.
Surfactant 16:0/14:0PC in Rat Models of Disturbed Alveolarization
As stated above, surfactant 16:0/14:0PC increased during the period of alveolarization in the rat. To determine the nature of this relationship, we examined BALF from two rat models of diminished alveolarization. The two models we used were postnatal exposure to either dexamethasone or 60% oxygen. Postnatal administration of dexamethasone to rats has been described to result in a reduced alveolarization (17, 39eC41). We choose to use a high-dose short-term treatment strategy, which results, at Day 10, in a significant decreased parenchymal complexity with larger and fewer alveoli compared with control subjects (17). Several studies have shown that neonatal hyperoxia of mice and rats also results in diminished alveolarization (18, 42eC44). Exposure of neonatal rats to 60% oxygen resulted in a significant reduction of total PC in BALF at Postnatal Day 10 (Figure 4a). By Postnatal Day 14, total PC values were no longer significantly different between 60% oxygen- and air-exposed animals (Figure 4e). Similar observations of reduced surfactant PC have been reported for neonatal rabbits exposed to 98% oxygen (45), although this effect could be the result of acute cellular injury. The content of individual PC molecules, including 16:0/16:0PC, 16:0/14:0PC, and 16:0/16:1PC, were also reduced in BALF of rats exposed to 60% oxygen (Figures 4beC4h). The overall reduction in surfactant PC synthesis may be caused by several factors. Hyperoxia is known to induce the release of a number of cytokines (46). Some of these cytokines—in particular, tumor necrosis factor —have been shown to reduce the activity of the rate-limiting enzyme in PC synthesis, CTP:phosphocholine cytidylyltransferase (47eC50). In addition, oxidant stress has been shown to affect another crucial lipid-synthesizing enzyme, glycerol phosphate acyltransferase (51). Alternatively, hyperoxia increases lipid peroxidation in rat type II cells (52), and therefore, type II cells may be shunt their lipid production from surfactant synthesis to cellular membrane repair. In contrast to hyperoxia, postpartum dexamethasone treatment resulted in a significant increase of total PC content in BALF at Day 10 (Figure 4a). In particular, monounsaturated PC molecules, such as 16:0/16:1PC (Figure 4c) and 16:0/18:1 (data not shown), were elevated in BALF of dexamethasone-treated neonatal rats. Similar observations have been reported for liver PC of dexamethasone-treated rats (53). Saturated PC species, including 16:0/16:0PC, were also elevated in BALF of dexamethasone-treated neonatal rats (Figure 4b), with the exception of 16:0/14:0PC, which was significantly reduced (Figure 4d). Interestingly, 16:0/14:0PC (Figure 4h) was the only PC species (Figures 4eeC4g) significantly reduced after 14 days of 60% oxygen exposure. Thus, the two rat models of reduced alveolarization had contrasting effects on surfactant PC production (i.e., upregulation with dexamethasone and downregulation with 60% oxygen). The only consistency with respect to surfactant PC was a constant decrease in 16:0/14:0PC concentration. Taken together, the results suggest that surfactant 16:0/14:0PC concentrations relate to the alveolarization process not only during normal lung development but also in two different models of diminished alveolar formation.
Surfactant 16:0/14:0PC in Human Diseases of Disturbed Alveolar Architecture
To evaluate the three predominant species of PC in human surfactant, we gathered tracheal aspirates from intubated neonates and BALF from adults. Data were subdivided on the basis of pathology. Tracheal aspirates were obtained from infants with either respiratory distress syndrome or BPD. BALF was obtained from adult patients with emphysema or idiopathic pulmonary fibrosis, and from patients after lung transplantation with no pronounced alveolar complication. These samples were compared with BALF obtained from patients with lung cancer who had normal alveolar architecture. Samples of patients with emphysema and infants with BPD displayed significantly reduced surfactant 16:0/14:0PC levels compared with all other patient groups tested (Figure 5b). Dipalmitoyl-PC did not significantly differ between the patient groups (Figure 5a). Thus, the changes in 16:0/14:0PC content in the emphysemic and BPD groups are likely not the result of a general loss of surfactant. Emphysema is characterized by abnormal, permanent enlargement of airspaces distal from the terminal bronchi, whereas one of the hallmarks of BPD seen in premature infants is alveolar simplification (i.e., larger but fewer alveoli). Although the human samples, specifically of adults, are limited, the reduced 16:0/14:0PC content in lavage fluid of BPD and emphysema is in line with the rat models of reduced alveolarization. As such, the results suggest that surfactant 16:0/14:0PC content in humans also relates to distal airspace architecture of the lung.
Possible Role for 16:0/14:0 PC in Surfactant during Alveolarization
The main feature of alveolarization is the subdivision of the preexisting voluminous saccules by septation, which leads to smaller units (alveoli) and an increased total surface area. These units then have the potential to increase in size during growth of the animal. One of the implications of saccular subdivision is an increase in alveolar curvature. Two groups have reported alterations in alveolar diameter (14) and volume (54) during the period of alveolarization in the rat (14, 54, 55). When we plotted radii calculated from the reported alveolar dimensions during rat development against 16:0/14:0PC in rat BALF, a striking relationship between 16:0/14:0PC content and the curvature was found (Figure 6a).
To further investigate this relationship, we performed light-scattering analysis on liposomes formed from lipids extracted of BALF. Lipids in BALF of Day 10 neonatal rats, which have a high surfactant 16:0/14:0PC content (Figure 4d), formed liposomes with an average particle size of 9.0 ± 0.4 e. In contrast, lipids in BALF from dexamethasone- and 60% oxygeneCtreated rats, which have a lower surfactant 16:0/14:0PC content (Figure 4d), formed significantly larger liposomes (13.2 ± 0.7 and 12.6 ± 0.6 e, respectively). The BALF liposomes of 22-day-old rats (14 ± 0.7 e) were significantly larger than those of 10-day-old rats, consistent with the lower 16:0/14:0PC content (Figures 2c and 2d). When we plotted the radii of BALF liposomes against BALF 16:0/14:0PC content, a strong correlation (r2 = 0.998) was found (Figure 6b), suggesting that 16:0/14:0PC increases the curving capacity of surfactant lipids. This would explain a potential basis for the increased 16:0/14:0PC levels in surfactant of higher curved airspaces that occurs during alveolarization. However, some caution is warranted because we did not consider changes in other variables, including surfactant proteins B and C.
Intrinsic curvature in membranes can be influenced by small polar head lipids (56) or by asymmetric acyl chain length (57eC59). Freeze fracture analysis of PC has shown that 16:0/16:0PC liposomes have a threefold greater radius compared with 16:0/14:0PC liposomes (60). The acyl chains of 16:0/14:0PC will not have the same surface packing as 16:0/16:0PC at 37°C, but because of its distinct acyl packing characteristics, it may obtain high surface pressures when spread on a highly curved interface. Therefore, we hypothesize that 16:0/14:0PC improves surfactant function during secondary septation, which is associated with more curved surface areas.
Finally, we investigated whether type II cells control acyl composition of PC during alveolarization. Using laser-capture microscopy and mass spectral lipid analysis, we found that 16:0/14:0PC content of rat type II cells increased postnatally from Day 7, peaked at Days 12 through 14, and subsequently declined until Day 21 when adult levels were reached (Figure 6c). The similarity between cellular and BALF 16:0/14:0PC content during the alveolarization period suggests that the lipid changes in the BALF are caused by type II cells producing a different surfactant. How the type II cells sense architectural changes and produce acyl-specific PC during alveolarization remains to be elucidated.
This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org
These authors contributed equally to this study.
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