Secretory IgA is a component of rabbit lung surfactant

Secretory IgA is a major protein component of rabbit lung surfactant purified by NaBr density gradient centrifugation from endobronchial lavage and minced lung tissue. Secretory IgA was found in both surfactant and non-surfactant fractions obtained from endobronchial lung washings. By contrast in minced-lung washings, which are not contaminated with proteins from the upper respiratory tree, secretory IgA is prominent only in the surfactant fraction. These findings indicate that in rabbit lung secretory IgA is present in the alveoli and is intimately associated with the surfactant system.     

Changes in quantity, composition, and surface activity of alveolar surfactant at birth

We hypothesized that when the lung makes the transition from the fluid- to the air-filled state at birth, there are changes in physical and functional properties of the alveolar surfactant. To test this hypothesis, newborn rabbits were killed at different times in the first 24 h of life, their lungs lavaged with ice-cold saline, and the lavage fluid subfractionated by differential centrifugation. The phospholipid and protein content and composition and the kinetics of surface tension lowering of the subfractions were examined. We found that with the onset of breathing, shifts occur in the distribution of surfactant subfractions as a surfactant apoprotein-free phospholipid fraction is generated. The ratio of rapidly sedimentable apoprotein-rich to slowly sedimentable, apoprotein-free fractions decreases from 31 at birth to 4 at 24 h of life. Concurrently, rates of surface tension lowering by the subfractions increase with time. The results suggest that the adult pattern of pool sizes and surface activity of alveolar surfactant is not present at birth but evolves slowly over the 1st day of life.     

Isolation of Secretory IgA from a Lung Surfactant Fraction

Although dipalmitoyl lecithin, is the essential component of the pulmonary surfactant system that is invoked for alveolar stability, there is no explanation as yet for the origin and role of certain proteins that are found with the phospholipid in pulmonary washings. Aqueous lavages obtained from rabbit lung contain three major proteins, two of which are serum proteins (albumin 60%, γ-globulin 10%), and the third, protein “T” (20%), is described here as pulmonary secretory immunoglobulin A (sIgA). The protein is first recovered quantitatively in the surface activelipid -protein fraction from filtration of pulmonary lavage on Sephadex G-200. The protein is then isolated from the lipid either by filtration on SDS-Sephadex G-200 or by ethanol-ether precipitation. After reductive cleavage with either mercaptoethanol or dithiothreitol and alkylation with iodoacetamide, three protein peaks are obtained from SDS-sephadex G-200 separation. The products of reductive cleavage are analogous to the secretory component (MW～60,000), H-chain (MW～50,000), L-chain (MW～25,000), and J-piece (MW～28,000) of secretory IgA frm colostrum. In inmunodiffusion the lung protein and colostrum sIgA show striking identity lines, as do antibodies to rabbit colostrum and to the lung protein. Amino acid and carbohydrate analyses reveal some differences between the two secretory immunoglobulins. Although this protein has been found together with the phospholipid surfactant of the lung in vitro, the present structural study concludes that the protein is SIgA. Although concurrent immunof luorescence studies showed that this protein is in the alveolar lining layer, we cannot as yet conclude that it belongs to the surfactant system of the lung.     

Alterations in rat alveolar surfactant phospholipids and proteins induced by administration of chlorphentermine

Chlorphentermine is a cationic amphiphilic drug which produces a phospholipid storage disorder in rat lungs. Experiments were carried out to characterize changes in the composition of acellular alveolar lavage materials and to study possible mechanisms by which pulmonary surfactant phospholipidosis is produced by administration of the drug. Following ten daily injections of chlorphentermine (25 mg/kg body weight), there are 12.2- and 13.6-fold increases of pulmonary lavage total phospholipids and disaturated phosphatidylcholines (disaturated PC), respectively. In addition, there is a 2.8-fold increase in total protein and a 12.7-fold increase in the surfactant apoprotein group with molecular weights from 28,000 to 32,000. We measured incorporation of labeled palmitate, choline and glycerol into disaturated PC in type II cells and alveolar macrophages isolated from control and chlorphentermine-treated animals. The drug does not affect the incorporation of labeled substrates into disaturated PC in either cell type. However, in alveolar macrophages there is a decrease in the rate of intracellular degradation of recently synthesized disaturated PC in chlorphentermine-treated animals. The drug also inhibits the phospholipase-induced catabolism of rat surfactant disaturated PC which occurs during incubation of alveolar lavage fluid in vitro at 37 degrees C. When the lavage fluid is divided into subfractions by differential centrifugation, a larger percentage of the phospholipid is distributed in the less sedimentable subfractions in chlorphentermine-treated animals relative to controls, suggesting the accumulation of older surfactant materials. These results suggest that chlorphentermine-induced phospholipidosis of pulmonary surfactant materials is due to decreased rates of phospholipid degradation.     

Uptake of lung surfactant subfractions into lamellar bodies of adult rabbit lungs

The goals of this investigation were to determine whether subfractions of alveolar surfactant that have different physical and biochemical properties are preferentially taken up from the alveolar air space into lamellar bodies and to correlate the magnitude of the uptake with the properties of the fractions. Radiolabeled subfractions were obtained by differential centrifugation of lavage fluid from rabbits that had been intravenously injected with radioactive palmitate. The subfractions were P (pellet) 3 (1,000 g, 20 min), P4 (60,000 g, 60 min), P5 (100,000 g, 16 h). Subfractions were instilled into the lungs of anesthetized spontaneously breathing adult rabbits, and lavage and lamellar body fractions were isolated at later times. P3 and P4 were taken up to a larger extent than was P5 or liposomes prepared from a P4 lipid extract. The fractions that were preferentially taken up (P3 and P4) contained surfactant apoprotein (APO) 36, tubular myelin, multilamellar vesicles, and were rapidly adsorbed to an air-water interface. P3 also contained APO 10. These results demonstrate that different forms of surfactant are recycled at different rates and suggest that there is specificity in the recycling process.     

Secretion of surfactant by primary cultures of alveolar type II cells isolated from rats

Pulmonary surfactant conventionally is prepared from material obtained by endobronchial lavage. Although it has been assumed that the components of surfactant are secreted by alveolar type II cells, direct proof of this assumption has not been available. Furthermore, it is possible that the final material obtained by lavage has been modified after secretion or altered during the isolation procedure. It has been shown previously that type II cells, after 1 day in primary culture, secrete saturated phosphatidylcholine, one of the lipid components of surfactant. Because saturated phosphatidylcholine is not unique to surfactant and because type II cells in culture lose differentiated characteristics over the first several days in culture, it has not previously been established how closely the secretory products of cultures of type II cells resemble surfactant as obtained by endobronchial lavage. We therefore studied the morphologic, physical and chemical characteristics of the material that type II cells secrete under basal conditions and after stimulation with terbutaline or 12-O-tetradecanoyl-13-phorbol acetate. The secreted material resembled surfactant obtained by lavage; it was similar morphologically to the lamellar material and tubular myelin seen in the fluid-filled alveoli of fetal rats, it lowered surface tension to 5 mN per meter, and it contained the 72000 dalton apolipoprotein of surfactant (as measured by the 'rocket' immunoelectrophoresis technique). When cells were incubated for 22 h with [1-(14)C]acetate, the distribution of radioactivity in the secreted material was very similar to the phospholipid composition of rat surfactant. We conclude that the material secreted by alveolar type II cells after 1 day in primary culture is similar to surfactant obtained by endobronchial lavage.     

Isolation and characterization of proteins associated with the lung surfactant system

Rabbit lung washings and purified lung surfactant were delipidated without precipitation or loss of protein. This enabled effective study of the proteins by electrophoretic and immunoelectrophoretic techniques. The lung washings contained secretory immunoglobulin A and several serum proteins. The protein composition of purified lung surfactant was the same as the unfractionated lung washings confirming our previous study which indicated that there is no specific protein associated with surfactant phospholipids obtained by alveolar lavage with isotonic saline.     

Isolation and Characterization of Proteins Associated with the Lung Surfactant System

Rabbit lung washings and purified lung surfactant were delipidated without precipitation or loss of protein. This enabled effective study of the proteins by electrophoretic and immunoelectrophoretlc techniques. The lung washings contained secretory immunoglobulin A and several serum proteins. The protein composition of purified lung surfactant was the same as the unfractionated lung washings confirming our previous study which indicated that there is no specific protein associated with surfactant phospholipids obtained by alveolar lavage with isotonic saline.     

Histochemical characterization of an antigen specific for the great alveolar cell in the mouse lung

Summary Previous papers reported on a specific antigenic marker for the great alveolar (type-II) cell of the mouse lung and described its recognition by a specific rabbit antiadult mouse lung serum. In the present study light- and electron-microscopical immunohistochemistry on fixed mouse lung sections showed the presence of the marker on the alveolar surface. The antigenic determinants recognized by the antibody were further characterized by immunoblotting and immunoprecipitation studies after in vitro translation of mouse lung messenger RNA.Immunoblots of a surfactant-enriched pellet of a bronchoalveolar lavage fraction of mouse lung showed that the antibody reacted with surfactant-associated proteins having apparent molecular weights of about 27,000, 32,000, and 38,000 daltons in SDS gels. Immunoblots of mouse-lung homogenate revealed the presence of 27,000, 30,000, 39,000, and 41,000 dalton proteins, presumably also surfactant-associated proteins. Immunoprecipitation after in vitro translation of mouse-lung mRNA showed specific reactivity only with a 12,000 dalton polypeptide, a component of the cell marker we were unable to relate to surfactant. Our findings indicate that the 12,000 dalton component of the antigenic marker for the great alveolar cell is a polypeptide whose synthesis is a lung-specific process and that the immunoreaction of the larger and surfactant-associated components is due to post-translational modifications.     

Histochemical characterization of an antigen specific for the great alveolar cell in the mouse lung

Previous papers reported on a specific antigenic marker for the great alveolar (type-II) cell of the mouse lung and described its recognition by a specific rabbit anti-adult mouse lung serum. In the present study light- and electron-microscopical immunohistochemistry on fixed mouse lung sections showed the presence of the marker on the alveolar surface. The antigenic determinants recognized by the antibody were further characterized by immunoblotting and immunoprecipitation studies after in vitro translation of mouse lung messenger RNA. Immunoblots of a surfactant-enriched pellet of a bronchoalveolar lavage fraction of mouse lung showed that the antibody reacted with surfactant-associated proteins having apparent molecular weights of about 27,000, 32,000, and 38,000 daltons in SDS gels. Immunoblots of mouse-lung homogenate revealed the presence of 27,000, 30,000, 39,000, and 41,000 daltons proteins, presumably also surfactant-associated proteins. Immunoprecipitation after in vitro translation of mouse-lung mRNA showed specific reactivity only with a 12,000 dalton polypeptide, a component of the cell marker we were unable to relate to surfactant. Our findings indicate that the 12,000 dalton component of the antigenic marker for the great alveolar cell is a polypeptide whose synthesis is a lung-specific process and that the immunoreaction of the larger and surfactant-associated components is due to post-translational modifications.     

Altered surfactant function and metabolism in rabbits with acute lung injury

Lung injury was induced in rabbits with N-nitroso-N-methylurethane (NNNMU), and saturated phosphatidylcholine (Sat PC) pool sizes and phospholipid compositions were measured in alveolar wash subfractions isolated by differential centrifugation (large and small surfactant aggregates). Surfactant metabolism also was studied using intravascular and intratracheal radiolabels. Protein permeability, gas exchange, and compliance were significantly abnormal as lung injury progressed. At peak injury, there was a decrease in the large aggregate Sat PC pool size in alveolar wash accompanied by increased uptake of Sat PC from the air space and increased specific activity of both intravascular and intratracheal radiolabels in lamellar bodies. This was followed by a marked rise in the small aggregate pool size in the alveolar wash and increased secretion of Sat PC into the air spaces. Phospholipid compositions, total phospholipid-to-protein ratios, and in vivo functional studies using a preterm ventilated rabbit model were abnormal for both large and small aggregate surfactant fractions from the lung-injured rabbits. These studies characterize quantitative, qualitative, and functional changes of alveolar wash surfactant subfractions in NNNMU-injured lungs.     

Clearance of surfactant phosphatidylcholine from adult rabbit lungs

Rabbits were given various doses of rabbit surfactant and treatment doses of approximately 100 mg/kg body wt of calf surfactant and Surfactant TA by tracheal injection. The linear loss of radiolabeled phosphatidylcholine from the total lung (alveolar wash and lung tissue) was 3.1, 1.5, and 1.8%/h for rabbit surfactant, calf surfactant, and Surfactant TA, respectively. After 24 h only 6% rabbit, 19% calf, and 9.7% Surfactant TA phosphatidylcholine were recovered by alveolar wash, and alveolar macrophage fractions contained less than 1% of the injected labeled phosphatidylcholine. The loss of rabbit surfactant phosphatidylcholine 24 h after tracheal injection did not change for doses in the range of 0.5-70 mumol phosphatidylcholine per kilogram, indicating nonsaturable clearance pathways. Very little of the labeled rabbit surfactant phosphatidylcholine lost from the lungs could be recovered in other organs, and 90% of the recovered labeled phosphatidylcholine in the liver was unsaturated, implying de novo synthesis using precursors from degraded phosphatidylcholine. The surfactant did not change endogenous lung phosphatidylcholine synthesis or its secretion to the alveolus. There were no adverse effects of the surfactant treatments noted in healthy rabbits.     

Polyfunctional role of the alveolar brush cells in the rat lung

By means of scanning and transmission electron microscopy it was demonstrated that the number of vacuoles located in the apical part of cytoplasm in alveolar brush cells of the regenerating rat lung increases, hyperplasia of Golgi-complex takes place and the activation of the protein-synthetising apparatus is evident. The immature surfactant material (osmiophilic lamellar bodies) and secretory dense core vesicles were found in the cytoplasm of alveolar brush cells. Intramuscular injections of colchicin to rats (0.1 mg/100 g body weight) 6 times during 24 hours before decapitation does not influence the number, topography and structure of microfibrilla bundles contained in a sufficient amount by alveolar brush cells. At the same time a part of microvilli of alveolar brush cells undergoes destruction and resorption under the action of colchicin. The data on ultrastructural organization of alveolar brush cells show that they are able to fulfill several functions: absorptive, contractile and secretory.     

Structural characteristics and intermolecular organization of human pulmonary-surfactant-associated proteins.

The structural relationships and intermolecular organization among the proteins associated with pulmonary surfactant are largely unknown. We studied the pulmonary-surfactant-associated proteins in the bronchoalveolar lavage fluid obtained from a patient with the clinical syndrome of alveolar proteinosis. The major proteins with Mr values of 32,000-36,000 and 62,000 formed thiol-dependent complexes (Mr greater than 400,000) with intermolecular disulphide bonds present in the collgenase-sensitive domains of these proteins. In contrast, other proteins, which were collagenase-insensitive, formed thiol-dependent oligomers that were not covalently linked to the major proteins. The associations of these proteins in the surfactant of a normal individual were similar. By amino acid analysis, two-dimensional peptide mapping and bacterial-collagenase digestion the 32,000-36,000-Mr and 62,000-Mr proteins were nearly identical. Differences in CNBr cleavage products suggested that the larger of the proteins was formed by non-disulphide, covalent, cross-links in the collagenase-sensitive domains of the 32,000-36,000-Mr proteins. Thus the evidence suggested that the lipid-associated proteins of Mr 32,000-36, 000 contained both disulphide and non-disulphide cross-links in the collagen-like N-terminal region of the proteins and form higher-Mr complexes. This organization may support the three-dimensional conformation of surfactant in the alveolar space.     

Surfactant metabolism in surfactant-treated preterm ventilated lambs

Preterm lambs were delivered at 132 days gestational age, treated with 100 mg/kg radiolabeled natural sheep surfactant or Surfactant TA, and ventilated for times up to 24 h. Compared with an untreated group that developed respiratory failure by 5 h, both surfactant-treated groups had stable respiratory function to 24 h. Although only approximately 13% of the labeled surfactant phosphatidylcholine was recovered by alveolar wash at 24 h, there was no significant loss of the labeled phosphatidylcholine from the lungs. Labeled palmitic acid intravascularly injected at 1 h of age comparably labeled lung phosphatidylcholine in the three groups of lambs at 5 h; however, only approximately 0.5% of the labeled phosphatidylcholine was secreted to the air spaces of surfactant-treated lambs at 24 h. Labeled lysophosphatidylcholine given with the natural sheep surfactant was taken up by the lungs, converted to phosphatidylcholine with 30-40% efficiency, and resecreted to the air spaces, demonstrating recycling of a phospholipid. The large surfactant aggregates recovered from alveolar washes by centrifugation were surface active and contained approximately 76% of the air-space phosphatidylcholine in both surfactant-treated groups. Although clinical status was comparable, alveolar washes and surfactant subfractions from Surfactant TA-treated lambs had better surface properties than did sheep surfactant-treated lambs. These studies identified no detrimental effects of surfactant treatments on endogenous surfactant metabolism and indicated that the surfactants used for treatments were recycled by the preterm ventilated lamb lung.     

Intrapulmonary catabolism of surfactant-saturated phosphatidylcholine in rabbits

Intrapulmonary surfactant catabolism was investigated by use of a phospholipase A1- and A2-resistant analogue of dipalmitoylphosphatidylcholine (DPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPC ether). [14C]DPC ether, made into liposomes with [3H]DPC and associated with 32P-labeled rabbit surfactant, was given intratracheally to 1-kg rabbits, which were killed at preset times to 48 h. Recoveries of radiolabel as saturated phosphatidylcholine (Sat PC) isolated from alveolar wash (AW), postlavage lung homogenate (LH), and alveolar macrophages were measured. All groups had similar AW and LH Sat PC pool sizes, indicating no perturbation of endogenous Sat PC pools. Despite a nearly fivefold accumulation of [14C]DPC ether in the lung by 48 h (P less than 0.01), the three probes had similar alveolar clearance curves. Furthermore, the Sat PC reutilization efficiency (41.6%) and turnover time (5.9 h) calculated for DPC ether were not different from values for the DPC and rabbit surfactant. Of the DPC ether (0.7%) and DPC (9%) labels recovered as PC in organs outside the lung, greater than 85% was unsaturated, indicating de novo synthesis using precursors from degraded PC. DPC ether was a useful probe of intrapulmonary DPC catabolism, and after alveolar uptake there was no direct reentry of intact DPC from the catabolic compartment(s) into the secretory pathway.     

Effects of alveolar surfactant aggregates on T-lymphocyte proliferation

The effects of alveolar large aggregate (LA) and small aggregate (SA) surfactant subfractions isolated from healthy adult rats on mitogen-stimulated proliferative responses of human peripheral blood mononuclear cells (PBMC) was examined. Various concentrations of total surfactant suppressed proliferation of stimulated lymphocytes by up to 95% of mitogen-stimulated cells alone. LA subfractions of total surfactant had no effect on proliferation, whereas SA significantly enhanced the lymphocyte proliferation at lower concentrations (7.8 microg/ml) compared to mitogen-stimulated cells alone. Higher concentrations of SA (62.5 microg/ml) inhibited lymphocyte proliferation. This concentration-dependent effect of SA on proliferation of PBMC was also present when cells were stimulated with various lectins including anti-CD3, concanavalin A and phytohemagglutinin. Analysis of the supernatant of mitogen-stimulated cell cultures treated with inhibitory concentrations of SA showed decreased amounts of interleukin (IL)-2, compared to cells alone, which could be reversed by adding exogenous IL-2 to the cell cultures with the SA. These results suggest that alveolar surfactant subfractions have distinct functions within the alveoli, both biophysically and with respect to their effects on the host's immunomodulatory responses.     

Alteration of surfactant function due to protein leakage: special interaction with fibrin monomer

In isolated rabbit lungs standardized amounts of edema were induced. Stimulation with the Ca ionophore A23187, leukotriene C4, Pseudomonas aeruginosa cytotoxin and human serum (activated complement) all resulted in protein leakage into the alveolar space with no change in the total phospholipid content. The pressure-volume characteristics of the lungs and the characteristics of the lavage surfactant (Wilhelmy balance) were markedly altered, correlating to the lavage protein content. The surfactant alterations were reproduced by addition of perfusion fluid protein to control surfactant in vitro. All changes were far less expressed or even missing in isolated lungs developing the same amount of edema due to omittance of proteins from the perfusion liquid. Different proteins added to control surfactant in the Wilhelmy balance showed a marked rank order of potency in interfering with surfactant function: immunoglobulins G and M and elastin less than albumin less than fibrinogen less than fibrin monomers. The fibrin monomer effect was reproduced by addition of thrombin to a surfactant fibrinogen mixture and was partly reversed by subsequent incubation with plasmin. In conclusion, high-permeability edema induced by different means results in alterations of lung mechanics and surface activity of lavaged surfactant, presumably due to protein surfactant interaction. Among different proteins, fibrin monomers are especially effective in interfering with surfactant function.     

Comparative proteomic analysis of lung lamellar bodies and lysosome-related organelles

Pulmonary surfactant is a complex mixture of lipids and proteins that is essential for postnatal function. Surfactant is synthesized in alveolar type II cells and stored as multi-bilayer membranes in a specialized secretory lysosome-related organelle (LRO), known as the lamellar body (LB), prior to secretion into the alveolar airspaces. Few LB proteins have been identified and the mechanisms regulating formation and trafficking of this organelle are poorly understood. Lamellar bodies were isolated from rat lungs, separated into limiting membrane and core populations, fractionated by SDS-PAGE and proteins identified by nanoLC-tandem mass spectrometry. In total 562 proteins were identified, significantly extending a previous study that identified 44 proteins in rat lung LB. The lung LB proteome reflects the dynamic interaction of this organelle with the biosynthetic, secretory and endocytic pathways of the type II epithelial cell. Comparison with other LRO proteomes indicated that 60% of LB proteins were detected in one or more of 8 other proteomes, confirming classification of the LB as a LRO. Remarkably the LB shared 37.8% of its proteins with the melanosome but only 9.9% with lamellar bodies from the skin. Of the 229 proteins not detected in other LRO proteomes, a subset of 34 proteins was enriched in lung relative to other tissues. Proteins with lipid-related functions comprised a significant proportion of the LB unique subset, consistent with the major function of this organelle in the organization, storage and secretion of surfactant lipid. The lung LB proteome will facilitate identification of molecular pathways involved in LB biogenesis, surfactant homeostasis and disease pathogenesis.     

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.