Clearance and recycling of pulmonary surfactant.

Clearance and recycling

of pulmonary

surfactant

JO RAE WRIGHT Cardiovascular Research Institute and Department of Physiology, University of California, San Francisco, California 94143

WRIGHT, JO RAE. Clearance and recycling of pulmonary surfactant. Am. J. Physiol. 259 (Lung Cell. Mol. Physiol. 3): Ll-L12, 1990.-In a steady state the rate of secretion of pulmonary surfactant lipids and proteins into the alveolar airspace must be balanced by the rate of removal. Several potential pathways for clearance have been identified including uptake by alveolar type II cells, which also synthesize and secrete surfactant components, uptake by other epithelial cells, and internalization by alveolar macrophages. A small amount of surfactant moves up the airways and through the epithelium-endothelium barrier into the blood. Some of the surfactant lipids and proteins that are cleared from the alveolar airspace appear to be “recycled” in that they appear in the lamellar body, a surfactant secretory granule found in the type II cell. Some surfactant lipids are degraded, probably intracellularly, and the degradation products are reutilized to synthesize new lipids. Several factors have been shown to affect internalization by the type II cell and/or alveolar clearance including the surfactant proteins, lipids, and known stimuli of surfactant secretion. Surfactant proteins may be involved in regulating pool size by modulating both secretion rates and uptake rates, possibly by a receptor-mediated process, although such receptors have not yet been identified or isolated. Clearance of surfactant lipids from the alveolar airspace is more rapid than clearance from the whole lung, and these two processes may be regulated by different factors. Elucidation of the factors that fine tune the balance between synthesis, secretion, and clearance of the lipid and protein components of surfactant awaits further investigation. alveolar lamellar

type II cell; surfactant body

phospholipids;

LITERATURE reveals a dramatic increase in the number of papers published on the subjects of clearance and recycling of surfactant in the past few years. The reasons for this increase are not known. One reason may be that several clinical trials are underway in which surfactant substitutes are being tested for the treatment of respiratory distress syndrome of the premature newborn, a disease that is associated with a lack of functional surfactant. All of these treatments involve administration of large doses of surfactant substitutes into the lungs of premature infants. Clearly, an understanding of clearance mechanisms is important in the design of a rational treatment regime. Another reason may be that many tools, such as purified surfactant proteins, recombinant surfactant proteins, synthetic peptides, antibodies against proteins, and fluorescentlabeled lipids and proteins, have recently become available and this has made possible studies that were not feasible a short time ago.

A SURVEY OF THE

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phospholipid

reutilization;

Although interest is increasing and our knowledge is expanding, there are still several gaps in our understanding of the mechanisms of clearance and recycling. The purpose of this article is to review the available information on the subject and to point out areas in which our knowledge is still incomplete. SURFACTANT

LIFE

CYCLE:

A BRIEF

OVERVIEW

Synthesis and Secretion of Surfactant Components

The data that are currently available suggest that most if not all of the components of surfactant are synthesized and secreted by the alveolar type II cell. The lamellar body of the type II cell has been regarded as an intracellular storage form of surfactant. This conclusion was based on the observation that the phospholipid composition of isolated lamellar bodies is virtually identical to that of surfactant obtained by bronchoalveolar lavage

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Society

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(reviewed in 8). It is not yet clear whether all three of the surfactant proteins are located in lamellar bodies. For a discussion of nomenclature of pulmonary surfactant associated proteins see Ref. 72. The major surfactant protein, SP-A, has been localized to lamellar bodies by immunohistochemistry (13, 40, 83, 87-89). SP-B has been identified in type II cells by immunohistochemistry at the light microscopic level (44). To date, there have been no published reports regarding the localization of SP-C. Thus the available evidence suggests that surfactant phospholipids and proteins are synthesized and secreted by the type II cell, although it is possible that other cells such as the Clara cell may also synthesize some of the components including SP-A (87). Electron-microscopic observations of fusion of the lamellar body-limiting membrane with the plasma membrane of the type II cell (43,79,89) support the idea that the lamellar body is not only a storage organelle but also a secretory granule. The stimuli involved in regulation of surfactant secretion were recently reviewed in this journal (8) and will not be discussed further here. Intra-Alveolar

Metabolism

of Surfactant

Lamellar bodies expand and form a complex latticelike structure called tubular myelin when they encounter the microenvironment of the alveolar airspace. The factors that trigger this event in vivo are not known but the conversion in vitro requires calcium (80). Indirect evidence suggests that tubular myelin is the precursor to the surface film (65), although results from some studies suggest that fractions of surfactant that are enriched in tubular myelin adsorb only slowly to an air-water interface (55, 85, 86). The mechanism by which the complex latticelike structure that consists of bilayers generates a monolayer is not known. It is generally agreed that the monolayer is enriched in dipalmitoylphosphatidylcholine (DPPC), which reduces surface tension at the air-liquid interface (reviewed in 93). The mechanism by which material exits from the monolayer is also not known. In addition to tubular myelin and the surface film, other forms of material that are presumably surfactant-related have been observed; these include large and small multilamellar vesicles, small unilamellar vesicles, and disklike structures thought to be open-ended bilayers (reviewed in 93). We and others (2,4,26,27,54,73) have suggested that surfactant undergoes metabolic transformations after it is secreted into the alveoli. Labeling studies, in which radioactive lipid precursors were injected intravenously into rabbits and the incorporation of radioactivity into subfractions of surfactant isolated by differential centrifugation was measured, suggested that “heavy” forms that contained tubular myelin were precursors to “lighter” forms that contained predominantly large and small vesicles. The “heavy” forms contained most of the surfactant proteins (91). A recent and interesting study by Gross and co-workers (26) suggested that similar pathways existed in mice, and they were able to simulate the conversion of heavy to light forms in vitro. The relationship of these potentially complex intraalveolar conversions to clearance and recvcling is not

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understood. There is some evidence, which will be discussed below, that fractions of surfactant that are enriched in various forms are cleared at different rates (94). However, because none of these forms have been purified to homogeneity, our understanding of their role in clearance or in intraalveolar metabolism is limited. It is interesting to speculate that some as yet unidentified factors, such as intraalveolar enzymes or ions, may be involved in regulation of extracellular metabolism and in targeting various components or forms of surfactant for specific clearance or degradation pathways. CLEARANCE

OF SURFACTANT

It seems that several possible metabolic fates await secreted surfactant lipids and proteins (Table 1). They could be recycled or reutilized. They could be degraded, either intracellularly or extracellularly, and the degradation products would be used for synthesis of new surfactant components. In this scenario, the molecules still remain associated with the surfactant system. In another scenario, the degradation products could be completely removed from the surfactant system and incorporated into other metabolic pathways, such as membrane lipids in other lung cells. Alternatively, the surfactant could be completely removed from the lung. Available evidence suggests that surfactant is cleared via all of these pathways to some extent, but the magnitude of clearance by each pathway probably varies considerably. Calculations of the actual rates of clearance by each of these pathways are complicated due to technical reasons that are discussed below. To date, most clearance studies have been done with surfactant lipid components. Relatively little is known about clearance of surfactant proteins. To simplify the review of the subject of clearance, it has been divided into two parts: clearance from the alveolar airspace and clearance from the whole lung. CLEARANCE

FROM

THE

ALVEOLAR

AIRSPACE

Uptake of Surfactant Components by Type II Cells

Results from several studies of different experimental designs suggest that the type II cell can internalize surfactant components and that the internalized material can be either recycled or degraded (Fig. 1). The idea that the type II cell may serve both as a source of newly synthesized surfactant as well as a vehicle for clearance is an intriguing one. Why would a cell go to the trouble of internalizing its own secretion? Clearly, there are large pools of lipid precursors that can be and are used to synthesize new material. It is possible that it is more TABLE

1. Theoretical pathways

for surfactant clearance

Uptake by alveolar type II cells alveolar macrophages alveolar type I cells airway or tracheal epithelial cells Movement up the airways and into esophagus Degradation in alveoli Movement through the epithelium/endothelium

into blood

or lymph


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FIG. 1. Theoretical pathways for surfactant clearance and metabolism by the alveolar type II cell. The type II cell synthesizes surfactant components which are then stored in lamellar body. Under the influence of appropriate stimuli the lamellar body is secreted into the alveolar airspace. The secreted surfactant undergoes complex alterations during its intra-alveolar metabolism including formation of a complex latt,icelike structure called tubular myelin. The form of surfactant that is taken up by the type II cell has not been identified. After surfactant lipids are internalized, some of them are degraded and products are either reutilized for synthesis of new surfactant lipids or the products may be removed from the type II cell and the surfactant life cycle. Some of the lipids can be reincorporated into the lamellar body and eventually resecreted. A role for surfactant proteins in enhancing lipid uptake has been demonstrated. Mechanisms that balance secretion, synthesis, and degradation are not understood.

“economical” in terms of cellular energy to recycle surfactant than to synthesize new material. It is also possible that degradation pathways are not very efficient and the material would b uild up if it were not recycled. Most of the studies regarding uptake of surfactant by type II cells fall into one of two categori .es: whole animal studies or studies with isolated type II cells. Radioactively labeled natural surfactant, which can be obtained by intravenous injection of a radioactive lipid precursor, or mixtures of radioactive surfactantlike lipids are instilled intratracheally into the lungs of animals or incubated with isolated type II cells. The association or uptake of the label i.nto whole lung tissue, isolated subcellular organelles, or isolated cells is then measured. The uptake or clearance of phosphatidylcholine has been most thoroughly studied because it is the major component of surfactant. Approximately 15 years ago, Geiger and co-workers (24) instilled radioactively labeled surfactant lipids into the lungs of rats and followed the fate of the radiolabel by autoradiography at the light microscope lev vel. Radioactive material appeared t !o be associated w ith both type I and type II cells 1 min after the instillation. However, by 2 h after the instillation, the number of labeled type I cells dropped by a factor of 2 and the number of labeled type II cells increased by a factor of 1.26. It was not possible to localize the label to specific intracellular organelles; however, this study did provide evidence that type II cells take up surfactant lipids. The results also suggested that some of the lipid was degraded, since radioactivity was detected in other compartments including liver, spleen, kidney, blood, and urine. Hallman and co-workers (28) provided convincing evi-

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dence that the type II cell not only internalizes surfactant lipids but that some of the lipids are incorporated into lamellar bodies. They instilled radioactively labeled surfactant lipids into the airspaces of adult rabbits and isolated fractions of subcellular organelles that were enriched in lamellar bodies. They observed that the radiolabeled lipid appeared rapidly in the lamellar body fractions. The decline in radioactivity of the lavage fluid was accompanied by a concomitant increase in lamellar body specific activity. Thus these results suggested that surfactant was not only taken up by type II cells, but that it was incorporated into lamellar bodies and resecreted. Morimoto and co-workers (60) also demonstrated that radioactively labeled DPPC that was instilled intratracheally into the lungs of adult rats appeared in lamellar body fractions. They also observed that some of the radioactive label appeared in other organs within five hours after the instillation. A similar experimental design was used by Jacobs and co-workers (32) to quantitate clearance and recycling in 3-day-old rabbits. Their data suggested that recycling was occurring at relatively fast rates. They estimated that the amount of phosphatidylcholine in the alveolar wash was -6.7 pmol and that -0.65 pmol/h of phosphatidylcholine moved from the alveolar wash into lamellar bodies. Based on these calculations, it appears that -10% of the alveolar pool is recycled per hour (Table 2). The hypothesis that surfactant is recycled is also supported by tracer studies in which radioactive lipid precursors are injected intravenously and the specific activities of lavage and lamellar body phospholipids are analyzed. Results from several investigations (e.g., 5, 28, 33, 38, 96) suggest that radiolabeled lipid remains in the surfactant system for several days. The specific activities in both lavage and lamellar bodies became approximately equal at long times after the pulse label and did not fall to zero even 100 h after the intravenous injection. These studies suggest that the lipid label shuttles between the extracellular and intracellular compartments. Chander and co-workers (7) first reported that isolated type II cells were capable of internalizing significant amounts of surfactant lipids. The uptake of lipid depended on the concentration of added lipid and on time and temperature. This observation paved the way for future experiments designed to study factors that regulate surfactant clearance. One of the advantages of studies with isolated type II cells is that the environment around the cell can be controlled. Thus it is possible to study the effects of specific proteins and lipids on the uptake process. In vivo studies are complicated by the fact that there is an endogenous pool of surfactant in contact with the type II cells and it is not possible to manipulate their environment as easily. However, the metabolic pathways in isolated cells may differ from those in the intact lung. It seems likely that important and different types of information will be obtained through both experimental designs. Efficiency of reutilization of phosphatidylcholine varies with age. The estimates of the percentage of reutilization

of phosphatidylcholine in adult animals varies with species, laboratory, and possibly experimental design. Esti-


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2. Calculated kinetic data for phosphatidylcholine turnover 3-Day-Old

Rabbits

Adult

Rabbits

Data are taken from studies by Pettenazzo and co-workers for adult rabbits (66) and for Jacobs a nd co-workers for 3-day-old rabbits (32). These results suggest that phosphatidylcholine (PC) turns over rapidly in adult and 3-day-old rabbits an d that the efficiency of reutilization is higher in adult animals than in 3-day-olds. For a discussion of the models and assum .ptions that apply to these calculations see Refs. 32 and 66. * Data were calculated from results reported in the study.

mates of the percentage of reutilization in adult rabbits range from 23% (35) to 85% (54). Gross and co-workers (25) estimated that the percentage of recycling in adult black mice was -85%. The percentage of reutilization may also vary with age. It has been estimated that 85% of the alveolar phosphatidylcholine is reutilized in 3-day-old rabbits (32). Jobe et al. (39) determined that the rate of uptake of phosphatidylcholine into lamellar bodies is slower in premature lambs than in older animals. However, the percentage reutilization in preterm lambs was not calculated in this study. The factors that determine the efficiency of reutilization are not known. Some factors, such as amounts of surfactant proteins, receptors for surfactant proteins, and degradative or synthetic enzymes, may be developmentally regulated and could be involved in determining the balance between degradation and reutilization. Clearance of lipids other thanphosphatidycholine. Most of the studies on lipid clearance have focused on the major surfactant associated lipid, phosphatidylcholine. However, the phospholipid composition of surfactant is complex and other phospholipids may play important roles in surfactant function. Jacobs and co-workers (37) concluded that the efficiency of reutilization of phosphatidylglycerol by adult rabbits is only 7% vs. a 23% efficiency of reutilization of phosphatidylcholine. Jacobs and co-workers (36) showed that phosphatidylethanolamine was reutilized less efficiently than phosphatidylcholine. Oyarzun and colleagues (64) found that liposomes containing phosphatidylglycerol were cleared more rapidly from the alveolar airspace than were liposomes that contained only DPPC. However, all studies of recycling are complicated by the fact that some phosphatidylcholine and phosphatidylglycerol are degraded and the degradation products are used for synthesis of new phospholipids. Lewis and co-workers (50) suggested that the catabolic pathways for saturated and unsaturated phosphatidylcholine are similar. Jacobs et al. (34) reported that four analogues of DPPC were cleared with equivalent rates and they concluded that the uptake process in 3-day-old rabbits for phosphatidylcholine was relatively nonspecific as long as the glycerol backbone contains no free hydroxyl group. Cholesterol is cleared from the alveoli with kinetics that are very similar to the kinetics of clearance of phosphatidylcholine (68). In summary, these studies suggest that the different

lipid components of surfactant may be cleared at different rates. The factors that determine the rate of clearance and fate of the different lipids are not known. Metabolic fate of surfactant internalized by type II cells.

Some of the lipid that is taken up by type II cells appears to be taken up intact, i.e., it is not degraded (7, 93). It seems likely that this lipid is resecreted, although this has not been proven. Results from several experimental approaches have provided evidence that a significant proportion of the lipid that is internalized is degraded. Isolated type II cells degrade internalized phosphatidylcholine and reutilize some of the products for synthesis of new lipid (7, 10). Similar conclusions were drawn by Fisher andco-workers (21, 22) from studies with an isolated perfused lung system. The metabolic fates of the other lipid components of surfactant have not been as thoroughly investigated. Lysophosphatidylcholine appears to be reutilized as phosphatidylcholine (34,37). The same group found that after instillation of [3H]phosphatidylglycerol, some tritium was found in phosphatidylcholine. Thus these studies also suggest that surfactant components can be degraded and reutilized for synthesis of new lipids. The factors that regulate the amount of lipid that is recycled or degraded are not known. Phosphatidylcholine taken up by type II cells in the presence of SP-A remained largely intact, whereas a larger proportion of the phosphatidylcholine taken up in the absence of SP-A was degraded (95). It is possible that SP-A (or other proteins) may direct lipids to different intracellular organelles, where they may either be recycled or degraded. However, the existence of such a directed transport is speculative. Chander and co-workers (10) reported that basic amines such as chloroquine and methylamine did not inhibit degradation. This fi .nding is consistent with the idea that degradation does not occur in an aci .dic compartment. Interestingly, most of the radioactivity associated with lamellar bodies was still in the form of DPPC, i.e., it had probably not been degraded. These results suggest that lamellar bodies that appear to have some properties of lysosomes, including an acidic pH (9) and lysosomal enzymes (29, 30), are not a major site of degradation of phosphatidylcholine. Additional studies will be required to characterize further the site of degradation and the factors that may be involved in regulating degradation. Uptake of surfactant proteins by type II cells. Relatively


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little is known about the uptake of surfactant proteins by type II cells. Young and co-workers (96, 97) demonstrated that 1251-labeled SP-A that was instilled into the trachea of adult rats as a lipid-SP-A complex appeared in lamellar bodies in a time-dependent fashion. Most of the lamellar body-associated protein migrated with the same molecular weight as the instilled material, and the authors concluded that there was little degradation of the internalized material. Some degradation did occur and labeled small peptides were associated with Golgi and microsome-enriched fractions. Snyder and co-workers (82) studied the clearance of 1251-labeled SP-A by neonatal rabbit lungs. The rabbits received an intratracheal injection of 1251-SP-A. Approximately 20% of the instilled radioactive label became associated with lung tissue by 1 h. Most of the rest of the radioactive label remained in the lavage. The time course of removal of 1251-SP-A from the lavage fluid was similar to that of [14C]DPPC. Most of the SP-A was not degraded over the time course of the experiment, although a small amount of radiolabeled, lowermolecular-weight protein (-16,000-18,000) was produced. Ryan and co-workers (78) provided evidence that SPA is taken up by isolated type II cells. They used the biotinyl ligand-avidin-gold technique to localize internalized SP-A to coated vesicles, endosomes, and multivesicular bodies. The metabolic fate of the internalized SP-A was not determined. A study of the turnover of surfactant proteins was published by King and co-workers (42) before specific antibodies were available to all of the surfactant proteins. They injected radioactive leucine as a precursor to surfactant proteins and at later times separated lavage proteins by electrophoresis and analyzed the specific activities of the proteins eluted from the gels. They suggested that proteins with molecular weights comparable to that of SP-A entered the alveoli with kinetics similar to the kinetics of DPPC and that low-molecularweight proteins were cleared with kinetics like those of phosphatidylcholine. Only a single study has been published thus far on the clearance of SP-C. Baritussio and co-workers (3) radiolabeled SP-C by intratracheal injection of [35S]methionine. Radiolabeled SP-C was mixed with 32P-labeled surfactant lipids and instilled into the tracheas of 3-dayold rabbits. Within 3 h, -50% of the instilled radiolabeled SP-C became inaccessible to lavage, whereas the amount of radioactive label that became associated with lung tissue had almost doubled. The authors concluded that the clearance of SP-C from the lavaged compartment was more rapid than the clearance of lipids. It was not determined whether the SP-C was taken up into lamellar bodies. Thus the available data to date suggest that SP-A and SP-C are cleared fairly rapidly from the alveolar airspace. One major unresolved issue is the rates at which these proteins are cleared. Specific activities were not measured in the studies performed with purified surfactant proteins. Therefore, it is impossible to quantitate their uptake into lamellar bodies or degradation. As more

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specific antibodies are generated and as techniques for separating and analyzing the proteins improve, these issues will very likely be resolved. There are no reported studies on the clearance of SP-B at this time. Mechanism of uptake by type II cells. The mechanism by which lipids and proteins are internalized by type II cells is not known. Lipid uptake by isolated type II cells is inhibited by metabolic inhibitors and by low temperature (7,93). These results suggest that energy is required for uptake. Williams (90) demonstrated the existence of an adsorptive endocytic pathway for uptake of lectins by type II cells. She found that the lectin from Maclura pomifera, which bound to the apical plasma membrane of the type II cell, was internalized and passed through pinocytic vesicles to multivesicular bodies and finally to lamellar bodies. Thus this study demonstrated that type II cells are capable of internalizing substances and incorporating them into lamellar bodies. However, it remains to be proven that this pathway is a route for transport of surfactant. Uptake by Other Epithelial Cells

It is reasonable to speculate that the type I cell, which covers -90% of the alveolar surface, may play a role in surfactant clearance. In fact, Geiger and co-workers (24) suggested that radioactivity became associated with type I cells immediately after the instillation of [3H]DPPC, although the resolution of the technique makes it difficult to determine whether label is actually associated with, or simply in close proximity to, the type I cell plasma membrane. To date, there are no published studies investigating the role of the isolated type I cell in clearance of either lipids or surfactant proteins. As the techniques for isolation of type I cells become more refined, it seems likely that our understanding of the role of the type I cell in surfactant clearance will be clarified. The role of tracheal and airway epithelial cells in clearance has not been investigated. It has been speculated that the nonciliated bronchiolar epithelial cell, also known as the Clara cell, may internalize surfactant. SPA has been localized by immunocytochemistry to the secretory granules (87) and the endoplasmic reticulum of the Clara cells (1,87). However, Phelps and colleagues (71) did not detect SP-A mRNA in human Clara cells by in situ hybridization. The reasons for this apparent discrepancy are not known. Additional studies will be required to determine whether Clara cells synthesize and/ or internalize SP-A and other surfactant associated proteins. Uptake of Surfactant Components by Alveolar Macrophages

The alveolar macrophage, an active phagocytic cell, is an attractive candidate for a route of surfactant clearance. It has been observed by electron microscopy that alveolar macrophages contain surfactant in the form of tubular myelin (62) and that macrophages take up surfactant lipids in vitro (15, 57, 93). SP-A has also been localized to phagocytic vacuoles (13, 83, 89) and SP-A had been shown to enhance the uptake of surfactantlike


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lipids by macrophages (95). In spite of these observations, several groups of investigators have suggested that the macrophage probably does not play a major role in surfactant clearance (5, 15, 66, 91). This suggestion is based on data from experiments in which animals received an intratracheal injection of radioactively labeled surfactant lipids or proteins, and macrophages were isolated and examined for radioactivity at various later times. It was found that the alveolar macrophages contained very little radioactivity. What could account for this apparent conflict? One explanation is that the macrophages do actively phagocytose surfactant lipids, but that the lipids are rapidly degraded and eliminated. Thus, although the uptake of lipids may be significant, the macrophage would contain little radioactive label. This idea is supported by a study by Miles and co-workers (57) who demonstrated that isolated alveolar macrophages degrade surfactant components. Young and co-workers (97) instilled 12?-SP-A into the lungs of spontaneously breathing adult rats and observed a rapid association of SP-A with the alveolar macrophage. Baritussio and co-workers (3) endogenously labeled SP-C with [35S]methionine and instilled the radiolabeled SP-C in a lipid complex into the lungs of 3-dayold rabbits and found that 24 h after the instillation, 5% of the radiolabeled SP-C was associated with alveolar macrophages. One potential problem with studies of uptake of surfactant by macrophages is that it is technically difficult to separate extracellular surfactant from the macrophages. Some surfactant cosediments with alveolar macrophages, making it difficult to distinguish intracellular from extracellular surfactant. Although the extracellular surfactant can be removed by washing of the cells, the repeated centrifugation may damage the macrophages and may result in release of internalized surfactant. It is not known if the clearance of surfactant by macrophages is altered by disease states. It is possible, for example, that macrophages may be activated in disease states and the phagocytosis of surfactant could be enhanced. It has been postulated that the disease of alveolar proteinosis, in which there is an intraalveolar accumulation of surfactant components, may be due to defective clearance by the macrophage. Proof of this hypothesis as well as further studies of the importance of the macrophage in both normal and disease states are required. Degradation Within Alveoli and Movement Through EpitheliumlEndothelium into Blood or Lymph Another possible route of surfactant clearance from the alveolar airspace is degradation within the alveoli and removal of the degradation products. There is some evidence that phospholipases can be found in lavage fluid. Miles and co-workers (56) reported that when lavage fluid from rat lungs is incubated at 37°C a significant proportion of the DPPC in the lavage fluid is degraded, and phospholipase activity has been detected in rat and human lavage material (81). In contrast, Lewis and colleagues (51) did not detect degradative activity in rat lung lavage fluid incubated in vitro at 37OC. No

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degradative activity was detected in lavage materials from rabbits, mice, or guinea pigs (56). Therefore, the data that are currently available do not allow us to definitely conclude that phospholipase activity is present or active in the alveolar subphase in vivo or whether it plays an important role in surfactant turnover. In fact, some data suggest that degradation of phospholipid is not a prerequisite for clearance. Oyarzun and co-workers (64) observed that the D-isomer of DPPC, which was not degraded by snake venom phospholipase A2 or by lung homogenate, was cleared at the same rate as L-isomer of DPPC. Jacobs and co-workers (34) instilled various analogues of DPPC into the lungs of 3day-old rabbits. They observed that L-CX-DPPC ether, which is resistant to hydrolysis, was reutilized to the same extent as was L-a-dipalmitoylphosphatidylcholine. They concluded that their results were most consistent with bulk uptake of surfactant from the alveolar airspace. Only a small amount of radioactive label appears in the lymph after intratracheal instillation of radioactively labeled lipids (14, 84). Thus it is generally agreed that this pathway is of minor importance for alveolar clearance, though it may be more significant for total lung clearance (discussed below). Movement

up the Airways

and into the Esophagus

Pettenazzo and co-workers (67) quantified the clearance of surfactant lipids up the airways. Approximately 7% of the administered dose was cleared via this route. Thus this seems to be a relatively minor pathway. The possibility that proteins may be cleared by this route has not been investigated. Phospholipid

Transfer

Proteins

and Clearance

It is well established that proteins isolated from lungs can catalyze the transfer of proteins between membranes (recently reviewed in 53). There are only a few studies that suggest that there may be transfer proteins in the alveolar subphase. Koumanov and colleagues (46) first provided evidence for the existence of such a protein and have subsequently obtained enriched preparations of the protein and shown that the preparations catalyze the transfer of both phosphatidylcholine and phosphatidylglycerol (45). Lumb and co-workers (52) reported that a protein fraction obtained from canine lavage catalyzed transfer of all major surfactant phospholipids and that the protein was probably not derived from serum. Whether these proteins function in vivo and whether they have sufficient activity to play a significant role in clearance remains to be established. What Regulates Alveolar

Clearance?

Estimates of secretion rates suggest that the pool of alveolar surfactant turns over very rapidly. It has been proposed that -lo-40% of the alveolar pool is secreted and cleared per hour (93). To achieve a steady state, the rate of secretion must be balanced by the rate of clearance. In addition, the synthesis of new material must be matched to balance degradation. How all of this is accom-


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plished is unclear. In fact, there is some controversy regarding how quickly pool size returns to normal after it is perturbed. Nicholas and co-workers (61) used swimming rats to study changes in pool size. Shortly after the onset of swimming, the alveolar pool size of rats increased from -7 to 9.5 mg phosphatidylcholine/g dry lung weight. Within 4 h after cessation of swimming the alveolar pool size had returned to approximately normal values. In contrast to these studies, Oguchi and coworkers (63) found that clearance of large doses of exogenously administered surfactant was quite slow. In these studies, adult rabbits received a dose of surfactant equivalent to approximately four times their normal alveolar pool size. Even by 72 h postadministration, the pool size had not returned to normal. Although the reasons for these differences are not known, many factors may influence the rate of clearance. Some of these factors are discussed below and are summarized in Table 3. Role of Surfactant Proteins in Regulating Surfactant Pool Size A paradigm for the role of surfactant proteins in regulating uptake of lipids exists in serum lipoprotein metabolism. Although our understanding of the role of surfactant proteins in regulating lipid uptake is not nearly as complete, evidence to date suggests that such a role exists. Studies by Claypool and co-workers (11, 12) provided evidence that an organic soluble fraction of surfactant, that contained the hydrophobic surfactant protein SP-C and some surfactant lipids, enhanced the uptake of liposomesby isolated type II cells. A recent study by Bates and co-workers (6) suggested that the phosphatidylglycerol in the organic solvent extract may be largely responsible for this enhancement. However, a recent study by Rice and co-workers (75) demonstrated that both natural and synthetic SP-B and SP-C enhance phosphatidylcholine uptake by type II cells. This stimulation did not seem to be cell specific, since both proteins also enhanced lipid uptake by lung fibroblasts. The enhancement was dependent on protein concentration, was not saturable, and occurred at both 4 and 37°C. SP-A also increases lipid uptake by isolated type II TABLE

3. Factors that affect surfactant clearance Factors

Surfactant proteins SP-A SP-B SP-c Ventilatory rates Surfactant secretatogues CAMP terbutaline ATP phorbol ester Phosphatidylglycerol

Reference

75,95 95 95 64 20-22

6, 64

Many different experimental approaches were used to test the effects of these factors on clearance and uptake. See text for details. These studies suggest that many factors may affect clearance rates. There is also suggestive evidence that other factors such as particle size, composition, manner of preparation of the surfactant, may affect the rates.

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L7

cells (75, 95). This effect was saturable and inhibited at low temperatures. SP-A did not significantly enhance lipid uptake by fibroblasts, although it did enhance uptake by isolated alveolar macrophages. These results as well as the observation that SP-A inhibits lipid secretion by isolated type II cells (18, 47, 74) are consistent with the possibility that type II cells have receptors for SP-A. Kuroki and co-workers (48) demonstrated that type II cells in primary culture bind SP-A with high affinity and that binding of 1251-SP-A is saturable. Ryan and colleagues (78) observed that binding of biotinylated SP-A to isolated type II cells was saturable. We observed that freshly isolated type II cells also bind SP-A with properties of a receptor-mediated process, although the affinity and number of receptors are different in the freshly isolated type II cells (92). Kuroki and co-workers (49) provided evidence that the receptor binding is probably associated with the inhibition of secretion, since factors that inhibited binding also abolished the inhibition of lipid secretion. It is not known whether the lipid uptake is mediated by the same or a different receptor, or by another mechanism altogether. Only indirect evidence is available to support the concept that surfactant proteins enhance lipid clearance in the intact animal. Subfractions of surfactant that contained SP-A were taken up from the alveolar airspace into lamellar bodies to a greater extent than were fractions of an organic solvent extract of surfactant (91). However, other studies suggest that liposomes that do not contain surfactant proteins are cleared at the same rate as natural surfactant that contains protein (35). The reasons for this difference are not known. Several factors could influence clearance including particle size, composition, whether the liposomes were mixed with natural surfactant before the instillation, the size of the dose, the propensity for the instilled label to bind to or exchange with the endogenous surfactant pool, and how the preparations were handled before the instillation (e.g., frozen, sonicated, vortexed, homogenized). Additional studies will be required to examine the effects of these variables and in particular the effects of specific surfactant proteins on uptake in the whole animal. Only a few studies have been done to examine the effects of surfactant administration on metabolism. Pettenazzo and co-workers (66) found that administration of large doses of surfactant (a dose of 100 mg/kg body weight, which is -IO-fold greater than the endogenous pool size) did not change endogenous lung phosphatidylcholine synthesis or secretion. Oguchi (63) and colleagues administered large doses of surfactant to 3-day-old rabbits and examined the effects of administration on the formation and secretion of labeled phosphatidylcholine. They determined that this dose of surfactant in this animal model had no effect on these parameters. Thus although studies with isolated cells suggest that surfactant components may inhibit secretion and synthesis of phosphatidylcholine, data from the whole animal studies do not support the concept that this is an important mechanism of regulation in vivo. However, the effects of surfactant might vary with composition, particle size,


L8

INVITED

animal age, and other variables. required to clarify this issue. Surfactant

Secretagogues

Enhance

Further studies will be

Lipid

Uptake

Fisher and co-workers (20-22) used an isolated perfused lung model to test the effects of surfactant secretagogues on clearance of phosphatidylcholine from the alveolar airspace. They found that isoproterenol, 8-bromoadenosine 3’,5’-cyclic monophosphate (8-Br-CAMP), terbutaline, adenosine triphosphate (ATP), or the phorbol ester, tetradecanoyl phorbol acetate (TPA) all enhanced clearance of radiolabeled phosphatidylcholine from the alveolar compartment. The secretagogues also enhanced degradation of the lipid. The effects of surfactant secretagogues on clearance have also been tested in a whole animal model. Pettenazzo (68) found that the ,&agonist metaproterenol sulfate enhanced the alveolar clearance of surfactant lipids. Interestingly, the drug had no effect on clearance of protein-free liposomes made from egg phosphatidylcholine, egg phosphatidylglycerol, cholesterol and a-tocopherol. Although there are several possible explanations for this difference, one intriguing possibility raised by the authors is that the liposomes and the surfactant lipids may be cleared by different pathways, which may be differentially sensitive to the P-agonist. Other Factors that Influence Ventilation and Phospholipid

Clearance: Composition

Oyarzun and co-workers (64) demonstrated that liposomes that contain phosphatidylglycerol and DPPC are cleared more rapidly from the airspace than are liposomes that contain only DPPC. Bates et al. (6) recently found that phosphatidylglycerol stimulated uptake of lipids by isolated type II cells. It is possible that the effect of phosphatidylglycerol is due to its negative charge (7), since liposomes containing either phosphatidylglycerol or phosphatidylserine were taken up to a greater extent than were liposomes that contained only phosphatidylcholine. Oyarzun et al. (64) also demonstrated that increasing the tidal volume of ventilated rabbits increases the clearance of lipids from the alveolar airspace. Ennema and co-workers (19) demonstrated that the flux of secretion of saturated phosphatidylcholine from the lamellar bodies into the alveolar lumen was increased in animals that were mechanically ventilated compared with animals that were breathing spontaneously. They speculated that ventilatory parameters may shift phospholipid metabolism from recycling to lysosomal degradation and result in an increased use of degradation products for the synthesis of new material. Further studies will be required to test this possibility. CLEARANCE

FROM

THE

WHOLE

LUNG

Another important aspect of surfactant metabolism is its time course of clearance from the whole lung. The general experimental approach for measuring whole lung clearance has been to instill radioactively labeled surfac-

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tant lipids into the lung and to measure the disappearance of radioactivity from the lavage fluid and from the whole lung tissue. Compared with alveolar clearance, clearance from the whole lung is slow. For example, Pettenazzo et al. (68) report that rabbit surfactant phosphatidylcholine is cleared from the whole lung at a rate of 56% per 24 hours, whereas the radiolabeled phosphatidylcholine was cleared from the alveolar wash at a rate of -90% per 24 hours. A critically important question, which is technically difficult to answer, is what percentage of the material that remains in the lungs is still associated with the surfactant system. Another unresolved issue is where all the cleared material goes. The data summarized in Table 4 indicate that excluding the lung, the liver contained the most (8%) of the recovered radioactivity. However, -43% of the instilled radioactivity could not be accounted for. It is possible that it is distributed in small amounts in other organs or lost from the body, perhaps as by-products of metabolism. Comparison of Clearance of “Tracer” Doses vs. Large Doses

Many clearance studies have been performed with small “tracer” doses of surfactant, usually -10% of the alveolar pool. However, the assumption that small doses act as tracers is potentially fraught with problems. For example, there are several morphological forms of surfactant found in the alveoli and there is some data that suggest that the protein composition of these forms may differ (91). We have no way of calculating the actual pool sizes of different forms of surfactant. Therefore, if a fraction of surfactant, enriched for example in tubular myelin, is used as a tracer, the amount of instilled material may actually represent a large proportion of the endogenous pool of that form. In fact, different fractions of surfactant are taken up into lamellar bodies at differ4. Percent recovery of radioactivity in various compartments 24 h after intratracheal instillation of radioactive phosphatidylcholine

TABLE

[3H]Choline Phosphatidylcholine

Compartment

Alveolar wash Lung tissue Macrophages Liver

Kidneys Spleen Bile Urine Serum Posterior

mediastinal

lymph

node

in

lOt2 34&l 1.7t0.4 8.2t0.5 1.8~0.1 0.1t0.04 Not detectable Not detectable 0.8tO.l 0.220.05

Values are means & SE and are taken from the work of Pettenazzo and co-workers (66). Adult rabbits received an intratracheal injection of [“HIcholine-labeled dipalmitoylphosphatidylcholine in the form of liposomes that had been premixed with natural rabbit surfactant. Twenty-four hours after the instillation, the radioactivity in various compartments was analyzed. The estimates of radioactivity in bile and urine are based on a sample from each. The estimate for alveolar macrophages was taken from the total cellular pellet from alveolar wash. These results show that most of the radioactivity that is recovered as phosphatidylcholine is in the lung. Relatively little is recovered in other organs. These results suggest that the phosphatidylcholine is being degraded.


INVITED

ent rates (94). Until it is possible to isolate the different forms of surfactant in more pure preparations, our ability to manipulate the system to gain accurate information will be limited. Several studies have also been done to characterize the clearance of large doses of surfactant in the range of 50150 mg/kg body weight. These doses are comparable to those used to treat premature infants with respiratory distress syndrome. The results from these studies indicate that the rate of clearance from the whole lung is relatively insensitive to the type of surfactant used. For example, Pettenazzo and co-workers (69) found that several types of surfactant (calf and sheep natural surfactant, TA-surfactant, and two aggregate sizes of lipid solvent extracted sheep surfactant) were all cleared from the lungs (alveolar wash plus lung tissue) of three-dayold rabbits at comparable rates. However, Ikegami et al. (31) found that liposomes of DPPC are cleared more rapidly from the whole lung than is natural surfactant. Interestingly, if the liposomes were allowed to first interact with natural surfactant prior to the instillation, they were cleared with kinetics that more closely resembled those of the clearance of natural surfactants. Thus these results suggest that interaction of instilled material with natural surfactant may affect clearance rates. These results also suggest that the factors that affect clearance of lipids from the whole lung differ from the factors that affect clearance from the alveolar airspace. For example, the fractions of surfactant that contain surfactant SP-A are taken up from the alveoli into lamellar bodies to a greater extent than are fractions that do not contain SP-A (94). However, preparations of surfactant that contain SP-A were cleared from the whole lung at rates that were very similar to those found for preparations that contained no detectable SP-A (63, 69). In addition, clearance from the whole lung does not appear to be saturable (70), whereas clearance from the alveolar airspace does appear to be saturable, at least in some experimental models (22). The Rate of Clearance from the Whole Lung Varies with Age

There appears to be very little catabolic activity in the lungs of preterm, ventilated lambs (39). Over the 24 h of the study, the percentage of recovered labeled phosphatidylcholine did not change significantly. However, the percentage of label that could be recovered in the lavage fluid decreased to 20% by 24 h, suggesting that there was rapid clearance of label from the alveoli. These results suggest that the level of degradative activity may vary with the age of the animal. CLEARANCE CONDITIONS

AND

RECYCLING

IN

PATHOLOGICAL

Whether clearance and recycling are altered by various diseases is unclear. In alveolar proteinosis excess surfactant is found in the airspaces; however, it is not known whether this is a disease caused by overproduction or defective clearance. One animal model in which there is an accumulation

L9

REVIEW

of surfactant

components

is the silica-exposed rat (16, and extracellular pools of surfactant lipids are increased (58). Kawada and co-workers (41) recently reported that SP-A content is increased in both lavage and type II cells without a change in SP-A mRNA levels. The possible mechanisms that could produce these altered pool sizes include increased synthetic rates, decreased catabolic rates, decreased clearance, increased number of type II cells, or some combination of these. Some evidence suggests that there is increased synthesis of phospholipids in lungs of silica-treated rats. Slices of silica-treated lungs had enhanced rates of synthesis of phosphatidylcholine (59, 77) and isolated hypertrophic type II cells from silica-treated lungs exhibited enhanced activities of phospholipid synthetic enzymes (41). However, it is also possible that precursor pool sizes or phospholipid turnover times are altered in these animals. Lewis and co-workers (51) found that the uptake of intratracheally instilled surfactant by the lung tissue and alveolar macrophages was not significantly different from control when corrected for cell number or lung weight. However, Dethloff and associates (l7), recently reported that the flux of phospholipid from the intracellular pool of silica-treated lungs was increased -13-fold and that the disappearance from the extracellular pool was increased only fivefold. Thus it is possible that silica treatment results in an imbalance of production, secretion, and clearance. The mechanisms by which these changes are brought about are not understood. 17, 23, 76, 77). Both the intracellular

SUMMARY

AND

CONCLUSIONS

Although it seems as though a great deal is known about clearance and recycling of surfactant components, there are still many unanswered questions. There is some indication that the turnover of surfactant is very rapid. This observation implies that the factors that regulate alveolar pool size must adapt rapidly in order to maintain steady-state conditions. There are also some indications that surfactant proteins may play important roles in regulating clearance, recycling, and pool size. The mechanisms by which these proteins act at the cellular level await identification. There is convincing evidence that the surfactant lipids can be both recycled and degraded. The signals that regulate the metabolic fate of the internalized lipids are not known, but one attractive hypothesis is that one or more of the surfactant proteins may be involved in targeting lipids to various intracellular organelles. The fates of the surfactant proteins are even less well understood. Another area that has received little attention is how the regulation of synthesis of new material is coordinated with the regulation of degradation and recycling. Currently, there is little evidence to suggest that treatment of premature babies with large doses of exogenous surfactant will have a negative impact on endogenous surfactant metabolism. However, our understanding of clearance of large doses is based primarily on studies conducted in term newborns or adult animals. Additional studies on clearance and metabolism in premature newborns, although technically difficult, will be vital. As new techniques, such as mutation of proteins


LlO

INVITED

and isolation of pure fractions of small populations of organelles and surfactant particles, are applied to the system, our understanding of the complex processes by which surfactant pool size is regulated will improve. The author thanks Deborah Cohen and Randy Decker for editorial assistance and preparation of the manuscript and Dr. John Clements for critical reading of the manuscript. This work was supported in part by National Heart, Lung, and Blood Institute Grants HL-30923 and HL-24075. J. R. Wright is an Established Investigator of the American Heart Association. Address for correspondence: J. R. Wright, Box 0130, CVRI, University of California, San Francisco, CA 94143. REFERENCES 1. BALIS, J. U., J. F. PATERSON, J. E. PACIGA, E. M. HALLER, AND S. A. SHELLEY. Distribution and subcellular localization of surfactant-associated glycoproteins in human lung. Lab. Inuest. 52: 657669,1985. 2. BARITUSSIO, A., L. BELLINA, R. CARRARO, A. ROSSI, G. ENZI, M. W. MAGOON, AND I. MUSSINI. Heterogeneity of alveolar surfactant in the rabbit: composition, morphology, and labelling of subfractions isolated by centrifugation of lung lavage. Eur. J. Clin. Inuest. 14: 24-29, 1984. 3. BARITUSSIO, A., A. BENEVENTO, A. PETTENAZZO, R. BRUNI, A. SANTUCCI, D. DALZOPPO, P. BARCAGLIONI, AND G. CREPALDI. The life cycle of a low-molecular-weight protein of surfactant (SPC) in 3-day-old rabbis. Biochim. Biophys. Acta 1006: 19-25, 1989. 4. BARITUSSIO, A., R. CARRARO, L. BELLINA, A. ROSSI, R. BRUNI, A. PETTENAZZO, AND G. ENZI. Turnover of phospholipids isolated from fractions of lung lavage fluid. J. AppZ. Physiol. 59: 1055-1060, 1985. 5. BARITUSSIO, A. G., M. W. MAGOON, J. GOERKE, AND J. A. CLEMENTS. Precursor-product relationship between rabbit type II cell lamellar bodies and alveolar surface-active material. Surfactant turnover time. Biochim. Biophys. Acta 666: 382-393,1981. 6. BATES, S. R., P. B. IBACH, AND A. B. FISHER. Phospholipids coisolated with rat surfactant protein C account for the apparent protein-enhanced uptake of liposomes into lung granular pneumocytes. Exp. Lung Res. 15: 695-708,1989. 7. CHANDER, A., W. D. CLAYPOOL, JR., J. F. STRAUSS, III, AND A. B. FISHER. Uptake of liposomal phosphatidylcholine by granular pneumocytes in primary culture. Am. J. Physiol. 245 (CeZZ. Physiol. 14): c397-C404,1983. 8. CHANDER, A., AND A. B. FISHER. Regulation of lung surfactant secretion. Am. J. Physiol. 258 (Lung CeZZ. MOL. Physiol. 2): L241L253,1990. 9. CHANDER, A., R. G. JOHNSON, J. REICHERTER, AND A. B. FISHER. Lung lamellar bodies maintain an acidic internal pH. J. BioZ. Chem. 261: 6126-6131,1986. 10. CHANDER, A., J. REICHERTER, ANY ,4. B. FISHER. Degradation of dipalmitoyl phosphatidylcholine by isolated rat granular pneumocytes and reutilization for surfactant synthesis. J. CZin. Inuest. 79: 1133-1138,1987. 11. CLAYPOOL, W. D., D. L. WANG, A. CHANDER, AND A. B. FISHER. An ethanol/ether soluble apoprotein from rat lung surfactant augments liposome uptake by isolated granular pneumocytes. J. CZin. Invest. 74: 677-684, 1984. 12. CLAYPOOL, W. D., D. L. WANG, A. CHANDER, AND A. B. FISHER. “Hydrophobic” surfactant apoproteins and augmentation of phospholipid recycling. Exp. Lung Res. 6: 215-222, 1984. 13. COALSON, J. J., V. T. WINTER, H. M. MARTIN, AND R. J. KING. Colloidal gold immunoultrastructural localization of rat surfactant. Am. Rev. Respir. Dis. 133: 230-237, 1986. 14. DAVIS, P. A., R. A. GUNTHER, AND C. E. CROSS. Clearance of instilled surfactant lipid from the lungs of unanesthetized sheep: lipids are differentially transported by nonlymphatic pathways. J. Lab. CZin. Med. 109: 191-200, 1987. 15. DESAI, R., T. D. TETLEY, C. G. CURTIS, G. M. POWELL, AND R. J. RICHARDS. Studies on the fate of pulmonary surfactant in the lung. Biochem. J. 176: 455-462, 1978. 16. DETHLOFF, L. A., L. B. GILMORE, AND G. E. R. HOOK. The relationshin between intra- and extra-cellular surfactant nhosnho-

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Regulation of pulmonary surfactant secretion and clearance.

Pulmonary clearance of 99mTc--DTPA and 99mTc-albumin in rabbits with surfactant dysfunction and lung injury.

Oleylethoxycarboxylate--an efficient surfactant for copper extraction and surfactant recycling via micellar enhanced ultrafiltration.

Pulmonary drug delivery by surfactant.

Inhibition of pulmonary surfactant function by phospholipases.

Biophysical inhibition of pulmonary surfactant function by polymeric nanoparticles: role of surfactant protein B and C.

Function and regulation of expression of pulmonary surfactant-associated proteins.

Pulmonary clearance of norepinephrine in lambs.

Pulmonary clearance of atrial natriuretic peptides.

Pulmonary corpora amylacea contain surfactant apoprotein.

Pulmonary surfactant: a surface chemistry viewpoint.

Characterization of pulmonary surfactant from ox, rabbit, rat and sheep.

Exogenous pulmonary surfactant as a vehicle for antimicrobials: assessment of surfactant-antibacterial interactions in vitro.

Rapid clearance of surfactant-associated palmitic acid from the lungs of developing and adult animals.

Pulmonary airway clearance mechanisms: a reappraisal.

Effect of amiloride and surfactant on lung liquid clearance in newborn rabbits.

Rapid clearance of xanthines from airway and pulmonary tissues.

Barrier or carrier? Pulmonary surfactant and drug delivery.

Characterization of antioxidant activities of pulmonary surfactant mixtures.

Inactivation of exogenous surfactant by pulmonary edema fluid.

The effects of porcine pulmonary surfactant on smoke inhalation injury.

Oxygen consumption of the newborn rabbit treated with pulmonary surfactant.

Increased expression of pulmonary surfactant proteins in oxygen-exposed rats.

Effects of activated polymorphonuclear leukocytes upon pulmonary surfactant in vitro.