Distinct effects of SP-B and SP-C on the uptake of surfactant-like liposomes by alveolar cells in vivo and in vitro

The effects of surfactant protein B (SP-B) and SP-C on the uptake of surfactant-like liposomes by alveolar type II cells and alveolar macrophages were studied both in vivo and in vitro. In vivo, mechanically ventilated rats were intratracheally instilled with fluorescently labeled liposomes that had SP-B and/or SP-C incorporated in different concentrations. Consequently, the alveolar cells were isolated, and cell-associated fluorescence was determined using flow cytometry. The results show that the incorporation of SP-B does not influence the uptake, and it also does not in the presence of essential cofactors. The inclusion of SP-C in the liposomes enhanced the alveolar type II cells at a SP-C to lipid ratio of 2:100. If divalent cations (calcium and magnesium) were present at physiological concentrations in the liposome suspension, uptake of liposomes by alveolar macrophages was also enhanced. In vitro, the incorporation of SP-B affected uptake only at a protein-to-lipid ratio of 8:100, whereas the inclusion of SP-C in the liposomes leads to an increased uptake at a protein-to-lipid ratio of 1:100. From these results, it can be concluded that SP-B is unlikely to affect uptake of surfactant, whereas SP-C in combination with divalent cations and other solutes are capable of increasing the uptake.     

Developmental regulation of re-uptake of phosphatidylcholine by type II alveolar epithelium

Type II alveolar epithelia produce, store and secrete pulmonary surfactant, a phospholipid and protein mixture which stabilizes alveoli at low lung volumes and, thereby, prevents alveolar collapse. We determined the developmental changes in the uptake, metabolism and reutilization of surfactant-related phospholipid in primary cultures of type II cells derived from fetal rat lung. Primary cultures of fetal and neonatal type II cells were incubated in media containing labelled liposomes. After the incubation phospholipids were extracted from the cells and uptake of label was analyzed. Re-uptake of radiolabelled dipalmitoyl phosphatidylcholine (DPPC) was concentration-dependent in undifferentiated fetal cells, differentiated fetal cells and neonatal cells. Re-uptake of DPPC by undifferentiated fetal cells was lower than re-uptake by both differentiated fetal and neonatal cells at 15 and 75 μM PC. Binding of DPPC to the cell surface involved a protein interaction, since trypsin was able to dissociate this trypsin-releasable fraction from internalized label. Undifferentiated fetal, differentiated fetal and neonatal cells all exhibited approx. 50% metabolic degradation of internalized phospholipid. Degraded lipids were reutilized in the synthesis of phosphatidylglycerol, but neonatal cells resynthesized twice as much phosphatidylglycerol as did undifferentiated fetal cells. These are the first studies which show that morphologically undifferentiated fetal type II cells are capable of the uptake of surfactant phospholipid as well as the degradation and reutilization of internalized phospholipid. Re-uptake, degradation and reutilization of internalized phospholipid appear to be under developmental control.     

In vivo and in vitro uptake of surfactant lipids by alveolar type II cells and macrophages

The uptake of fluorescent-labeled liposomes (with a surfactant-like composition) by alveolar macrophages and alveolar type II cells was studied using flow cytometry, in vivo by instillation of the labeled liposomes in the trachea of ventilated rats followed by isolation of the alveolar cells and determination of the cell-associated fluorescence, and in vitro by incubation of isolated alveolar cells with the fluorescent liposomes. The results show that the uptake of liposomes by the alveolar cells is time and concentration dependent. In vivo alveolar macrophages internalize more than three times as many liposomes as alveolar type II cells, whereas in vitro, the amount of internalized liposomes by these cells is approximately the same. In vitro, practically all the cells (70-75%) internalize liposomes, whereas in vivo only 30% of the alveolar type II cells ingest liposomes vs. 70% of the alveolar macrophages. These results indicate that in vivo, only a small subpopulation of alveolar type II cells is able to internalize surfactant liposomes.     

Surfactant peptides stimulate uptake of phosphatidylcholine by isolated cells

To determine whether small hydrophobic surfactant peptides (SP-B and SP-C) participate in recycling of pulmonary surfactant phospholipid, we determined the effect of these peptides on transfer of 3H- or 14C-labelled phosphatidylcholine from liposomes to isolated rat alveolar Type II cells and Chinese hamster lung fibroblasts. Both natural and synthetic SP-B and SP-C markedly stimulated phosphatidylcholine transfer to alveolar Type II cells and Chinese hamster lung fibroblasts in a dose- and time-dependent fashion. Effects of the peptides on phospholipid uptake were dose-dependent, but not saturable and occurred at both 4 and 37 degrees C. Uptake of labelled phospholipid into a lamellar body fraction prepared from Type II cells was augmented in the presence of SP-B. Neither SP-B nor SP-C augmented exchange of labelled plasma membrane phosphatidylcholine from isolated Type II cells or enhanced the release of surfactant phospholipid when compared to liposomes without SP-B or SP-C. Addition of native bovine SP-B and SP-C to the phospholipid vesicles perturbed the size and structure of the vesicles as determined by electron microscopy. To determine the structural elements responsible for the effect of the peptides on phospholipid uptake, fragments of SP-B were synthesized by solid-phase protein synthesis and their effects on phospholipid uptake assessed in Type II epithelial cells. SP-B (1-60) stimulated phospholipid uptake 7-fold. A smaller fragment of SP-B (15-60) was less active and the SP-B peptide (40-60) failed to augment phospholipid uptake significantly. Like SP-B and SP-C, surfactant-associated protein (SP-A) enhanced phospholipid uptake by Type II cells. However, SP-A failed to significantly stimulate phosphatidylcholine uptake by Chinese hamster lung fibroblasts. These studies demonstrate the independent activity of surfactant proteins SP-B and SP-C on the uptake of phospholipid by Type II epithelial cells and Chinese hamster lung fibroblasts in vitro.     

Role of clathrin- and actin-dependent endocytotic pathways in lung phospholipid uptake

We evaluated the contribution of endocytotic pathways to pulmonary uptake of surfactant lipids from the alveolar space. Resting and stimulated 8-bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP) uptake of unilamellar liposomes labeled with either [(3)H]dipalmitoylphosphatidylcholine ([(3)H]DPPC) or 1-palmitoyl-2-[12-(7-nitro-2-1,3-benzoxadiazol-4-yl) amino] dodecanoyl-phosphatidylcholine (NBD-PC) was studied in isolated perfused rat lungs and isolated type II cells. Amantadine and phenylarsine oxide, inhibitors of clathrin-mediated endocytosis, each decreased [(3)H]DPPC uptake under resting conditions by approximately 40%; their combination had no additional effect. Cytochalasin D, an inhibitor of actin-dependent processes, reduced liposome uptake by 55% and potentiated the effect of either clathrin inhibitor alone. Relative inhibition for all agents was higher in the presence of 8-Br-cAMP. The effect of inhibitors was similar for liposomes labeled with [(3)H]DPPC or NBD-PC. By fluorescence microscopy, NBD-PC taken up by lungs was localized primarily to alveolar type II cells and was localized to lamellar bodies in both lungs and isolated cells. These studies indicate that both clathrin-mediated and actin-mediated pathways are responsible for endocytosis of DPPC-labeled liposomes by alveolar type II cells in the intact lung.     

Association of chlorphentermine with phospholipids in rat alveolar lavage materials, alveolar macrophages and type II cells

Administration of chlorphentermine to rats leads to an increase in the phospholipid content of pulmonary surfactant materials and alveolar macrophages. It is known that this drug binds to pure phospholipids and prevents their degradation by phospholipases. Therefore, experiments were carried out to determine if chlorphentermine binds to surfactant phospholipids in vitro and to measure the in vivo association of drug with phospholipids in alveolar lavage materials from rats injected with [14C]chlorphentermine. The presence of chlorphentermine in alveolar macrophages, type II cells and other small pneumocytes (a population of lung cells which does not include alveolar macrophages or type II cells) from treated animals was also assessed. Binding of the drug to surfactant phospholipids, as measured with the fluorescent probe, 1-anilino-8-naphthalene sulfonate, occurs in vitro and does not differ in various subfractions of alveolar lavage materials isolated by differential centrifugation. Following daily administration of chlorphentermine to rats for 3 days, the drug appears to be associated with surfactant phospholipids such that the molar ratio is 1:100 (chlorphentermine/phospholipid). Chlorphentermine is also associated with alveolar macrophages (molar ratio, 1:18) and type II cells (molar ratio, 1:33). Not much drug is associated with the population of other lung cells (molar ratio, 1:333). In alveolar macrophages, approx. 70% of the drug seems to be bound to phospholipid and/or sequestered in subcellular organelles. However, only 20% of the chlorphentermine is bound and/or sequestered in type II cells. The results of these experiments suggest that following chlorphentermine administration, the drug is associated with phospholipids in acellular pulmonary lavage materials, alveolar macrophages and type II cells. This drug-phospholipid interaction may impair phospholipid degradation and lead to a phospholipidosis in surfactant materials and alveolar macrophages.     

Macrophage and type II cell catabolism of SP-A and saturated phosphatidylcholine in mouse lungs

Type II cells and macrophages are the major cells involved in the alveolar clearance and catabolism of surfactant. We measured type II cell and macrophage contributions to the catabolism of saturated phosphatidylcholine and surfactant protein A (SP-A) in mice. We used intratracheally administered SP-A labeled with residualizing (125)I-dilactitol-tyramine, radiolabeled dipalmitoylphosphatidylcholine ([(3)H]DPPC), and its degradation-resistant analog [(14)C]DPPC-ether. At 15 min and 7, 19, 29, and 48 h after intratracheal injection, the mice were killed; alveolar lavage was then performed to recover macrophages and surfactant. Type II cells and macrophages not recovered by the lavage were subsequently isolated by enzymatic digestion of the lung. Radioactivity was measured in total lung, lavage fluid macrophages, alveolar washes, type II cells, and lung digest macrophages. Approximately equal amounts of (125)I-dilactitol-tyramine-SP-A and [(14)C]DPPC-ether associated with the macrophages (lavage fluid plus lung digest) and type II cells when corrected for the efficiency of type II cell isolation. Eighty percent of the macrophage-associated radiolabel was recovered from lung digest macrophages. We conclude that macrophages and type II cells contribute equally to saturated phosphatidylcholine and SP-A catabolism in mice.     

Immunocytochemical localization of the major surfactant apoproteins in type II cells, Clara cells, and alveolar macrophages of rat lung

The adsorptive properties of phospholipids of pulmonary surfactant are markedly influenced by the presence of three related proteins (26-38 KD, reduced) found in purified surfactant. Whether these proteins are pre-assembled with lipids before secretion is uncertain but would be expected for a lipoprotein secretion. We performed indirect immunocytochemistry on frozen thin sections of rat lung to identify cells and intracellular organelles that contain these proteins. The three proteins, purified from lavaged surfactant, were used to generate antisera in rabbits. Immunoblotting of rat surfactant showed that the IgG reacted with the three proteins and a 55-60 KD band which may be a polymer of the lower MW species. Specific gold labeling occurred over alveolar type II cells, bronchiolar Clara cells, alveolar macrophages, and tubular myelin. In type II cells labeling occurred in synthetic organelles and lamellar bodies, which contain surfactant lipids. Lamellar body labeling was increased fivefold by pre-treating tissue sections with a detergent. Multivesicular bodies and some small apical vesicles in type II cells were also labeled. Secondary lysosomes of alveolar macrophages were immunoreactive. Labeling in Clara cells exceeded that of type II cells, with prominent labeling in secretory granules, Golgi apparatus, and endoplasmic reticulum. These observations clarify the organelles and pathways utilized in the elaboration of surfactant. After synthesis, the proteins move, probably via multivesicular bodies, to lamellar bodies. Both lipids and proteins are present in tubular myelin. Immunologically identical or closely similar proteins are synthesized by Clara cells and secreted from granules which appear not to contain lipid. The role of these proteins in bronchiolar function is unknown.     

Binding and uptake of surfactant protein D by freshly isolated rat alveolar type II cells

Alveolar type II cells secrete, internalize, and recycle pulmonary surfactant, a lipid and protein complex that increases alveolar compliance and participates in pulmonary host defense. Surfactant protein (SP) D, a collagenous C-type lectin, has recently been described as a modulator of surfactant homeostasis. Mice lacking SP-D accumulate surfactant in their alveoli and type II cell lamellar bodies, organelles adapted for recycling and secretion of surfactant. The goal of current study was to characterize the interaction of SP-D with rat type II cells. Type II cells bound SP-D in a concentration-, time-, temperature-, and calcium-dependent manner. However, SP-D binding did not alter type II cell surfactant lipid uptake. Type II cells internalized SP-D into lamellar bodies and degraded a fraction of the SP-D pool. Our results also indicated that SP-D binding sites on type II cells may differ from those on alveolar macrophages. We conclude that, in vitro, type II cells bind and recycle SP-D to lamellar bodies, but SP-D may not directly modulate surfactant uptake by type II cells.     

Surfactant protein D influences surfactant ultrastructure and uptake by alveolar type II cells

Surfactant protein D (SP-D) is a member of the collectin family of the innate host defense proteins. In the lung, SP-D is expressed primarily by type II cells. Gene-targeted SP-D-deficient [SP-D(-/-)] mice have three- to fivefold higher surfactant lipid pool sizes. However, surfactant synthesis and secretion by type II cells and catabolism by alveolar macrophages are normal in SP-D(-/-) mice. Therefore, we hypothesized that SP-D might regulate surfactant homeostasis by influencing surfactant structure, thereby altering its uptake by type II cells. Large (LA) and small aggregate (SA) surfactant were isolated from bronchoalveolar lavage fluid (BALF) from SP-D(-/-), wild-type [SP-D(+/+)], and transgenic mice in which SP-D was expressed under conditional control of doxycycline in alveolar type II cells. Uptake of both LA and SA isolated from SP-D(-/-) mice by normal type II cells was decreased. Abnormally dense lipid forms were observed by electron microscopy of LA from SP-D(-/-) mice. SA from SP-D(-/-) mice consisted of atypical multilamellated small vesicles. Abnormalities in surfactant uptake by type II cells and in surfactant ultrastructure were corrected by conditional expression of SP-D in vivo. Preincubation of BALF from SP-D(-/-) mice with SP-D changed surfactant ultrastructure to be similar to that of SP-D(+/+) mice in vitro. The rapid changes in surfactant structure, increased uptake by type II cells, and decreased pool sizes normally occurring in the postnatal period were not seen in SP-D(-/-) mice. SP-D regulates uptake and catabolism by type II cells and influences the ultrastructure of surfactant in the alveolus.     

Binding of adenovirus capsid to dipalmitoyl phosphatidylcholine provides a novel pathway for virus entry

Adenovirus (Ad) is an airborne, nonenveloped virus infecting respiratory epithelium. To study the mechanism of Ad entry, we used alveolar adenocarcinoma A549 cells, which have retained the ability of alveolar epithelial type II cells to synthesize the major component of pulmonary surfactant, disaturated phosphatidylcholine. Stimulation of phosphatidylcholine secretion by calcium ionophore or phorbol ester augmented the susceptibility of these cells to Ad. Both Ad infection and recombinant-Ad-mediated transfection increased in the presence of dipalmitoyl phosphatidylcholine (DPPC) liposomes in culture medium. Importantly, in the presence of DPPC liposomes, virus penetrates the cells independently of virus-specific protein receptors. DPPC vesicles bind Ad and are efficiently incorporated by A549 lung cells, serving as a virus vehicle during Ad penetration. To identify the viral protein(s) mediating Ad binding, a flotation of liposomes preincubated with structural viral proteins was employed, showing that the only Ad protein bound to DPPC vesicles was a hexon. The hexon preserved its phospholipid-binding properties upon purification, confirming its involvement in virus binding to the phospholipid. Given that disaturated phosphatidylcholine not only covers the inner surface of alveoli in the lungs but also reenters alveolar epithelium during lung surfactant turnover, Ad binding to this phospholipid may provide a pathway for virus entry into alveolar epithelium in vivo.     

Metabolism of phosphatidylglycerol by alveolar macrophages in vitro

In whole animal studies, it has been shown that turnover of surfactant dipalmitoylphosphatidylglycerol (DPPG) is faster than that of dipalmitoylphosphatidylcholine (DPPC). The goal of this investigation was to characterize the metabolism of DPPG by alveolar macrophages and to determine whether they contribute to the faster alveolar clearance of DPPG. Isolated rat alveolar macrophages were incubated with liposomes colabeled with [(3)H]DPPG and [(14)C]DPPC. Macrophages internalized both lipids in a time- and temperature-dependent manner. The uptake of both lipids was increased by surfactant protein (SP) A and by adherence of the macrophages to plastic slides. The isotope ratio of DPPC to DPPG internalized by macrophages in suspension in the absence of SP-A was significantly lower than the isotope ratio in liposomes, suggesting that macrophages preferentially internalize DPPG when SP-A is absent. Phospholipase activity in macrophage homogenate was higher toward sn-2-labeled DPPG than toward sn-2-labeled DPPC. These studies show that alveolar macrophages play an important role in catabolizing surfactant lipids and may be partially responsible for the relatively faster clearance of DPPG from the lung.     

Protective effects of elevated endogenous surfactant pools to injurious mechanical ventilation

Depletion of alveolar macrophages (AM) leads to an increase in endogenous surfactant that lasts several days beyond the repletion of AM. Furthermore, impairment to the endogenous pulmonary surfactant system contributes to ventilation-induced lung injury. The objective of the current study was to determine whether increased endogenous surfactant pools induced via AM depletion was protective against ventilation-induced lung injury. Adult rats were intratracheally instilled with either control or dichloromethylene diphosphonic acid (DMDP) containing liposomes to deplete AMs and thereby increase endogenous surfactant pools. Either 3 or 7 days following instillation, rats were exposed to 2 h of injurious ventilation using either an ex vivo or in vivo ventilation protocol and were compared with nonventilated controls. The measured outcomes were oxygenation, lung compliance, lavage protein, and inflammatory cytokine concentrations. Compared with controls, the DMDP-treated animals had significantly reduced AM numbers and increased surfactant pools 3 days after instillation. Seven days after instillation, AM numbers had returned to normal, but surfactant pools were still elevated. DMDP-treated animals at both time points exhibited protection against ventilation-induced lung injury, which included superior physiological parameters, lower protein leakage, and lower inflammatory mediator release into the air space, compared with animals not receiving DMDP. It is concluded that DMDP-liposome administration protects against ventilation-induced lung injury. This effect appears to be due to the presence of elevated endogenous surfactant pools.     

The Lung Surfactant and Immune System Response to Intratracheal Administration of “Empty” Liposomes

Abstract

After intratracheal administration of “empty” lecithin-cholesterol liposomes to rats it was found out twofold enhancement of the surfactant content with maximum on the 2nd-3rd day and with normalization to the control level by the 7th day. Phagocytic index of the alveolar macrophages was also increased. It was shown the change of the blast-transformation reaction of bronchoalveolar lavage and blood lymphocytes. Immune complexes content in bronchoalveolar lavage at different period of time after liposomes administration increased 1.5-2-fold. The natural killers (NK) activity of cells obtained from bronhoalveolar lavage and blood was enhanced 10 times and 2 times respectively. It is supposed that enhancement of lung surfactant phospholipid content is caused by substrate stimulation of type II alveolocytes activity. The stimulation of immunocompetent cells might be connected with imitation of bacterial attack by liposomes with proteins adsorbed on their surface.     

Catalase immunocytochemistry allows automatic detection of lung type II alveolar cells

In mammalian lung, type II pneumocytes are especially critical in normal alveolar functioning, as they are the major source of surfactant and the progenitors of type I alveolar cells. Moreover, they undergo proliferation and transformation into type I cells in most types of cellular injury, where flattened type I pneumocytes are selectively destroyed. Hyperplasia of alveolar type II cells has also been described in some human chronic lung diseases. In lung, type II pneumocytes and non-ciliated bronchiolar cells are the unique cell types that contain a considerable amount of peroxisomes. Due to the presence of dihydroxyacetone phosphate acyltransferase and non-specific lipid-transfer protein, these organelles have been suggested to be involved in the synthesis and/or transport of the lipid moiety of surfactant. In the present research, the peroxisomal marker enzyme catalase was immunolocalised at the light microscopic level, utilising the avidin-biotin complex method, in lung specimens excised from newborn, adult and aged rats. In all the examined stages the immunoreactivity was so selective for type II pneumocytes it allowed quantitation of these cells by an automated detection system. This was accomplished on specimens from newborn rat lung, in which labelled alveolar cells were counted by a grey level-based procedure and their main morphometric parameters were determined.     

Effects of pulmonary surfactant and surfactant protein A on phagocytosis of fractionated alveolar macrophages: relationship to starvation.

Pulmonary surfactant isolated from bronchoalveolar lavage fluid of rat lung contained a high content of surfactant protein A (SP-A) in starved for 2 days compared to fed controls, but this phenomena returned to baseline following more than 4 days starvation. As determined by immunoperoxidase staining of lung sections using SP-A antibody, SP-A could be consistently observed in nonciliated bronchiolar (Clara) cells, alveolar type II cells and some alveolar macrophages (AM). Fc receptor-mediated phagocytosis of AM was enhanced by SP-A, which was dependent on the dosis and reached a maximum at 10 micrograms of SP-A/ml. Antibody to SP-A completely inhibited the enhanced response of phagocytosis. When exposed AM subpopulations, separated into four fractions (I, II, III and IV) by discontinuous Percoll gradient, to SP-A or pulmonary surfactant prepared from rats fed and starved for 2 days enhanced their phagocytic activity in high dense cells (III and IV), particularly to SP-A and pulmonary surfactant from rats starved for 2 days. Whereas little change in lower dense fractions (I and II) were seen in all exposures except for SP-A that enhanced the cells of fraction II. These results supported the concept that pulmonary surfactant and its apoprotein, SP-A, are a factor to regulate lung defense system including activation of AM that undergo different processes following starvation.     

Identification of beta-adrenergic receptors in pulmonary alveolar type II cells

Specific beta-adrenergic receptors have been identified in dissociated preparations of rabbit lung cells greatly enriched for alveolar type II cells and compared with receptors in preparations of mixed lung cells and erythrocytes. Freshly isolated type II cells as well as mixed dissociated lung cells and erythrocytes from fetal (28 days gestation) and adult rabbits contained high-affinity, low-capacity binding sites for [3H]dihydroalprenolol (DHA). Binding to all preparations was stereospecific and characteristic of the beta 1-subtype of beta-adrenergic receptors. The concentrations of the receptors were similar in mixed lung cells and alveolar type II cells, indicating that beta-adrenergic receptors are present not only in type II cells but also in other lung cell types. When the contribution of erythrocytes to receptor concentration observed in type II cells was determined, it was found to be insignificant. In mixed lung cells, both the affinity and concentration of the receptors were higher in adult than fetal preparations. The affinity of the receptors was also higher in adult than fetal type II cells, although we did not find a significant age-related difference in receptor concentrations in this cell type. These results suggest that stimulation of surfactant secretion observed after exposure of lung tissue to beta-adrenergic agonists is mediated by specific beta-adrenergic receptors on alveolar type II cells.     

In vitro fusion of lung lamellar bodies and plasma membrane is augmented by lung synexin.

Lamellar bodies of lung epithelial type II cells undergo fusion with plasma membrane prior to exocytosis of surfactant into the alveolar lumen. Since synexin from adrenal glands promotes aggregation and fusion of chromaffin granules, we purified synexin-like proteins from bovine lung cytosolic fraction, and evaluated their effect on the fusion of isolated lamellar bodies and plasma membrane fractions. Synexin activity, which co-purified with an approx. 47 kDa protein (pI 6.8), was assessed by following calcium-dependent aggregation of liposomes prepared from a mixture of phosphatidylcholine:phosphatidylserine (PC:PS, 3:1, mol/mol). Lung synexin caused aggregation of liposomes approximating lung surfactant lipid-like composition, isolated lamellar bodies, or isolated plasma membrane fraction. Lung synexin promoted fusion only in the presence of calcium. It augmented fusion between lamellar bodies and plasma membranes, lamellar bodies and liposomes, or between two populations of liposomes. However, selectivity with regard to synexin-mediated fusion was observed as synexin did not promote fusion between plasma membrane and liposomes, or between liposomes of surfactant lipid-like composition and other liposomes. These observations support a role for lung synexin in membrane fusion between the plasma membrane and lamellar bodies during exocytosis of lung surfactant, and suggest that such fusion is dependent on composition of interacting membranes.     

Platelet-activating factor (PAF)-acetylhydrolase and PAF-like compounds in the lung: effects of hyperoxia.

Platelet-activating factor (PAF)-acetylhydrolase is the enzyme modulating in tissues and biological fluids the concentration of the proinflammatory factors PAF and PAF-like oxidation products of phospholipids (PAF-like compounds). We investigated whether there is a relation between PAF-acetylhydrolase activity and the concentration of PAF-like compounds in bronchoalveolar lavage (BAL). We found that alveolar type II cells are an additional source of PAF-acetylhydrolase in BAL beside macrophages. Secretion of PAF-acetylhydrolase was stimulated by phorbol ester in alveolar type II cells but not in macrophages. Studies in BAL suggested that secreted PAF-acetylhydrolase was bound to alveolar surfactant. Exposure of rats to high oxygen concentration reduced the activity of PAF-acetylhydrolase in BAL and macrophages, but not in plasma or alveolar type II cells. In contrast, hyperoxia increased the concentration of PAF-like-compounds, lipid hydroperoxides and malonedialdehyde in plasma but not in BAL. Therefore, we conclude that neither the oxidant-induced decrease of the PAF-acetylhydrolase activity nor the direct peroxidation of surfactant lipids in the alveoli provide a likely mechanism for hyperoxia-induced lung injury. Instead, lung injury is apparently caused by lipid peroxidation in plasma rather than by high oxygen pressure in the alveoli.     

Surfactant apoprotein Mr = 26,000-36,000 enhances uptake of liposomes by type II cells

The alveolar type II cell which synthesizes and secretes surfactant also plays a major role in the reuptake of surfactant lipids. In a recent in vivo study we found that the subfractions of natural surfactant that contained the surfactant protein with molecular weights of 26,000-36,000 (SP-26-36) were preferentially taken up into lamellar bodies of type II cells to a greater extent than were fractions that did not contain SP-26-36. Because the subfractions of natural surfactant in that study differed in other properties than the presence or absence of SP-26-36, the current study was undertaken to determine whether purified SP-26-36 enhanced the uptake of surfactant-like lipids by freshly isolated type II cells. SP-26-36 increased the uptake of label in radioactive surfactant-like lipids by up to 10-fold, and the effect of SP-26-36 was dependent on time, protein concentration, and temperature. The enhancement was inhibited by heat-treating the protein, by a polyclonal antibody against SP-26-36, and by metabolic inhibitors. The distribution of radioactivity in cell-associated phospholipids differed if cells were incubated with or without SP-26-36. If SP-26-36 was present during the incubation, greater than 96% of the radioactivity remained associated with phosphatidylcholine. In the absence of SP-26-36, only 85% of the radioactivity remained associated with phosphatidylcholine and 7% of the label appeared in phosphatidylglycerol. We hypothesize that SP-26-36 may act as a ligand to direct surfactant lipids to type II cells, perhaps to different metabolic pathways, and to regulate recycling and surfactant homeostasis.