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An albumin-associated PLA2-like activity inactivates surfactant phosphatidylcholine secreted from fetal type II pneumocytes Jolanta E. Damas and Max H. Cake

Am J Physiol Lung Cell Mol Physiol 301:L966-L974, 2011. First published 9 September 2011; doi: 10.1152/ajplung.00103.2011 You might find this additional info useful... This article cites 69 articles, 18 of which you can access for free at: http://ajplung.physiology.org/content/301/6/L966.full#ref-list-1 Updated information and services including high resolution figures, can be found at: http://ajplung.physiology.org/content/301/6/L966.full

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American Journal of Physiology - Lung Cellular and Molecular Physiology publishes original research covering the broad scope of molecular, cellular, and integrative aspects of normal and abnormal function of cells and components of the respiratory system. It is published 12 times a year (monthly) by the American Physiological Society, 9650 Rockville Pike, Bethesda MD 20814-3991. Copyright © 2011 the American Physiological Society. ISSN: 1040-0605, ESSN: 1522-1504. Visit our website at http://www.the-aps.org/.

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Am J Physiol Lung Cell Mol Physiol 301: L966–L974, 2011. First published September 9, 2011; doi:10.1152/ajplung.00103.2011.

An albumin-associated PLA2-like activity inactivates surfactant phosphatidylcholine secreted from fetal type II pneumocytes Jolanta E. Damas and Max H. Cake School of Biological Sciences and Biotechnology, Murdoch University, Murdoch, Western Australia, Australia Submitted 31 March 2011; accepted in final form 5 September 2011

surfactant phospholipids; phosphatidylcholine deacylation; lyso-phosphatidylcholine

for normal lung function. Through its ability to adsorb rapidly to the alveolar surface and reduce surface tension, surfactant increases lung compliance, thereby reducing the work of breathing (19, 50, 52). The ability of surfactant films to attain very low surface tension during compression stabilizes lung alveoli at end expiration by preventing alveolar collapse. The essential role of pulmonary surfactant in normal lung function is illustrated by the proven efficacy of exogenous surfactant to act as a replacement in the treatment of neonatal respiratory distress syndrome (NRDS) (8, 20, 33, 62). Acute respiratory distress syndrome (ARDS), which can affect both adults and children, has a more complicated pathology than the simple absence of surfactant but shares many of the symptoms of NRDS, such as diminished lung compliance, a marked reduction in effective lung volume and profound hypoxemia (23). However, clinical trials with the most effective formulations of synthetic surfactant, which had been used successfully in the treatment of NRDS, showed that PULMONARY SURFACTANT IS ESSENTIAL

Address for reprint requests and other correspondence: M. H. Cake, School of Biological Sciences and Biotechnology, Faculty of Science and Engineering, Murdoch Univ., Murdoch, Western Australia, 6150, Australia (e-mail: [email protected]). L966

these formulations had only a modest and transient beneficial effect when administered to ARDS patients (45). Extracted bronchial fluid (lavage) from ARDS patients has been consistently shown to have elevated levels of serum proteins (47), and the ratio of soluble proteins to lung surfactant in lavage correlates closely with the severity and clinical outcome of patients suffering from ARDS (24). Lavage from ARDS patients also has a markedly reduced activity of surfactant as reflected in the speed with which the material adsorbs to the air-water interface and the minimum surface tension achieved (47). Biophysical studies of lung surfactant deliberately mixed with serum proteins show that, at sufficiently high concentrations of proteins, an ARDS-like depression of lung surfactant activity can be obtained (27). Plasma contains an array of protein and nonprotein components, which act with different potencies as inhibitors of surfactant function. These include fibrinogen, hemoglobin, albumin, cholesterol, platelet-activating factor, neutrophils (18, 28). There are two mechanisms by which these agents operate: 1) physical interactions, which result in competition between plasma proteins and surfactant phospholipids for space at the air-liquid interface during the process of adsorption and thus prevent the phospholipids from forming a film at the interface (26), and 2) chemical interactions that include effects on both the lipids and the proteins (2, 68). As an example, phospholipase A2 (PLA2), which is present in plasma and hydrolyzes phosphatidylcholine (PC) to the inactive lyso-phosphatidylcholine (lyso-PC) form, has been implicated in the pathogenesis of a variety of pulmonary diseases including ARDS (4, 13). The potential importance of an elevated PLA2 in the pathophysiology of ARDS is supported by the observation that intratracheal administration of PLA2 can induce lung injury, including alveolar edema, accumulation of inflammatory cells, and thickening of the alveolar wall (6, 13), which are pathological features typically seen in the lungs of ARDS patients. In this study, cultured fetal type II pneumocytes were used as a model to examine the effects of human serum on inactivation of secreted phospholipids. As in previous studies, serum was shown to enhance the conversion of PC to lyso-PC, so a range of techniques were employed to identify the component responsible for this deacylation of surfactant lipids and to compare its properties with those of secretory PLA2. The results show that the serum component was not secretory PLA2 and that its activity comigrated with albumin. Moreover, exposure of secreted phospholipids to albumin [including recombinant human serum albumin (rHSA)] causes an elevation in the level of lyso-PC, suggesting that PLA2 is an intrinsic activity of albumin. We therefore propose that this particular mechanism of surfactant inactivation needs to be considered prior to any attempt to develop improved procedures for the treatment of ARDS.

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Damas JE, Cake MH. An albumin-associated PLA2-like activity inactivates surfactant phosphatidylcholine secreted from fetal type II pneumocytes. Am J Physiol Lung Cell Mol Physiol 301: L966–L974, 2011. First published September 9, 2011; doi:10.1152/ajplung.00103.2011.—Type II pneumocytes are responsible for the synthesis and secretion of pulmonary surfactant, which reduces surface tension in lung alveoli, thus decreasing their tendency to collapse during expiration. For this effect to be sustained, the integrity of the surface-active components of surfactant must be maintained. This study has shown that, when cultured type II pneumocytes are exposed to lipoprotein-free serum (LFS), the level of lyso-phosphatidylcholine (lyso-PC) in the secreted surfactant phospholipids is markedly elevated with a concomitant decline in the level of phosphatidylcholine (PC). This effect is the result of hydrolysis of surfactant PC by a phospholipase A2 (PLA2)like activity present within serum. Anion-exchange chromatography, gel filtration chromatography and preparative electrophoresis of human LFS have shown that this PLA2-like activity coelutes with albumin and is biochemically distinct from the secretory form of PLA 2 . Furthermore, specific inhibitors of PLA 2 such as pbromophenacyl bromide, aristolochic acid, and palmitoyl trifluoromethyl ketone do not inhibit this activity of serum. Commercially purified human serum albumin fraction V and recombinant human serum albumin (rHSA) are almost as effective as LFS in enhancing the level of lyso-PC in the media. The latter finding implies that rHSA directly generates lyso-PC from secreted PC and suggests that this PLA2-like activity may be an intrinsic attribute of albumin.

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% secretion ⫽

media dpm media dpm ⫹ cellular dpm



100 1

Composition of secreted lipids. Secreted radiolabeled phospholipids were analyzed by thin-layer chromatography on Whatman silica gel LK6DF plates (Whatman International, Maidstone, UK), focusing on the choline-containing lipids PC, lyso-PC, and sphingomyelin. Prior to the application of each sample, the plates were activated in an oven at 110°C for 1 h. The plate was then placed into a chromatographic chamber lined with Whatman 3MM paper wetted with the developing solvent, CHCl3-CH3OH-7 M NH4OH (65:35:5 vol/vol/ vol), as described by Finkelstein et al. (16). After 10 min, chromatography was initiated and continued until the solvent front was 0.5–1.0 cm from the top of the plate. Dried plates were then exposed to iodine vapor, phospholipids were identified by use of purified standards, and the labeled phospholipids were isolated by scraping the silica gel directly into a Teflon glass homogenizer and subsequently extracting it with 3 ml of CHCl3-CH3OH-formic acid-water (19.4:19.4: 0.4:0.8 vol/vol/vol/vol), according to the method of Gross et al. (21). Chromatographic characterization of the PLA2-like activity of LFS HiTrap Heparin-Sepharose chromatography. Aliquots of either lipoprotein-free serum (LFS; 250 ␮l) or secretory PLA2 (sPLA2; 160 units), previously exposed to heat treatment at 60°C for 30 min, were applied to a HiTrap Heparin-Sepharose column (1 ⫻ 5 cm) equilibrated with 20 mM NH4HCO3, pH 7.5. The column was washed with 40 ml of this buffer at a flow rate of 60 ml/h prior to eluting any bound material with a 60 ml 0 –3.0 M NaCl gradient in the same buffer. DEAE-Sepharose chromatography. Aliquots (2.5 ml) of either LFS or 1,600 units of sPLA2, previously heat treated (as above), were applied to a CL-6B DEAE-Sepharose column (2.7 ⫻ 45 cm) equilibrated with 25 mM KCl in 20 mM NH4HCO3, pH 7.5. The column was run at a constant flow rate of 15 ml/h, and eighty 4-ml fractions were collected. An additional 140 fractions were collected after the application of a linear gradient of KCl in 20 mM NH4HCO3, pH 7.5.

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Animals. Nineteen-day pregnant rats of the Wistar albino strain of Rattus norvegicus were used in all experiments. These inbred animals have a gestation period of 22 days and were supplied by the Animal Resource Centre, Murdoch, Western Australia. Male and female rats were caged together overnight, and conception was considered to occur on the following morning when a vaginal smear detected the presence of sperm. This was designated day 0 of pregnancy and is accurate to within ⫾8 h. All experiments complied with the National Health and Medical Research Council guidelines and were approved by the Murdoch University Animal Ethics Committee. Materials. The chemicals and materials used in the experiments are as follows: collagenase A (Clostridium histolyticum) (Boehringer Mannheim, Mannheim, Germany); Eagle’s minimal essential medium (MEM) and newborn calf serum (NBCS) (Trace Biosciences, NSW, Australia); penicillin G, streptomycin sulfate, lipoprotein-free human serum, solubilized ␤-amphotericin (Fungizone), arachidonic acid, human serum albumin fraction V (HSA V), very low-density lipoprotein (VLDL), low-density lipoprotein (LDL), high-density lipoprotein (HDL), PLA2 from porcine pancreas, EGTA, bromophenacyl bromide (Sigma Chemical, St. Louis, MO); Optiphase “HiSafe” II scintillant (FSA Laboratories, Leicester, UK); PD-10, DEAE CL-6B, and HiTrap Heparin Sepharose columns (Amersham Pharmacia Biotech, NSW, Australia); chloramphenicol (NBL Gene Sciences, Northumberland, UK); L-glutamine (GIBCO Laboratories, Chagrin Falls, OH); palmitoyl trifluoromethyl ketone and methyl arachidonyl fluorophosphonate (Calbiochem-Novabiochem, NSW, Australia); rHSA [mammalian-cell derived: ProspecTany Technogene, Israel; Pichia pastoris (yeast)-derived: Albumin Bioscience, Huntsville, AL]; 1,2 bis-heptanoyl thioglycero-phosphocholine (Sapphire Bioscience, NSW, Australia); L-3-phosphatidylcholine 1,2-di[1-14C] palmitoyl (54 mCi/ mmol), and [methyl-3H]choline chloride (81 Ci/mmol) (Radiochemical Centre, Amersham, Buckinghamshire, UK). Preparation of materials for cell culture. NBCS was treated with acid-washed charcoal to remove endogenous steroids, as previously described (58), sterilized with use of a 0.22-␮m GS filter, and either stored at ⫺20°C or used immediately in the preparation of culture medium. Rat IgG was purified according to the method of Hudson and Hay (30). The protein concentration of the solution was adjusted to 1 mg/ml prior to subjecting it to lyophilization and storage at ⫺20°C. Powdered MEM was reconstituted and supplemented with NaHCO3, L-glutamine, and penicillin G/streptomycin to final concentrations of 0.2%, 2.6 mM, and 100 IU/100 ␮g/ml, respectively, and the medium was adjusted to pH 7.4. After filter sterilization, Fungizone was added to a final concentration of 3.23 ␮g/ml. To yield complete medium, charcoal-treated NBCS was added to a final concentration of 10%. Isolation and culture of fetal type II pneumocytes. Alveolar type II pneumocytes were isolated from 19-day fetal rats by tissue dissociation with collagenase (58) and partially purified by the procedure of Dobbs et al. (12). The cells were resuspended in NBCS-supplemented medium and plated onto Falcon Primaria tissue culture plates. Cultures were maintained in a humidified atmosphere of 5% CO2-95% air at 37°C in a Forma Scientific (model 3250) water-jacketed CO2 incubator. Unless otherwise specified, the type II cell cultures were given an initial media change 24 h after plating and thereafter at 48-h intervals. After 72 h in culture, the plates were fully confluent and consisted predominantly of type II pneumocytes. Assay of secretion of surfactant phospholipids. After 72 h in culture, the serum-supplemented MEM was removed and the cells were washed with 2 ml prewarmed balanced salts solution (BSS; 137 mM NaCl, 2.7 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 6 mM NaHCO3, 0.15 mM NaH2PO4, 1.35 mM Na2HPO4, 5.55 mM D-glucose, pH 7.4) and then incubated for 24 h at 37°C with serum-free MEM containing [methyl-3H]choline chloride (1 ␮Ci/ml). After this incubation, labeled

medium was removed and the cells were washed three times with 2 ml of BSS and allowed to equilibrate with 1.7 ml serum-free MEM for 1 h. After equilibration, two plates, which represent secretion at time zero, were removed from the incubator and placed on ice. The medium was removed immediately and the cells washed three times with 0.83 ml of BSS. Control plates were established by adding 34.5 ␮l of sterile water (the vehicle) to two plates. The remaining plates received an equivalent volume of the agent to be examined. Both control and test plates were incubated for a further 3 h, before being placed on ice. The medium was removed and the cells washed as described above. The combined media and washings from each of the equilibrium, control, and test plates were vortexed briefly and centrifuged for 2 min at 1,000 g at 4°C to sediment any detached cells. The top 3.5 ml of each tube was removed and used for lipid extraction. The cells from the plates were removed from each plate by scraping with a polyethylene scraper into 0.8 ml of water and 1.0 ml of methanol, followed by a further scraping with 1.0 ml of methanol. Extraction of lipids from media and cells. After the addition of 50 ␮l of L-3-phosphatidylcholine 1,2-di[1-14C] palmitoyl ([14C]DPPC) (⬃10,000 dpm), as a recovery standard, and 20 ␮l (2.5 mg/ml L-␣-phosphatidylcholine), as a carrier, to each 3.5 ml of collected media and to the cell extracts, the lipids were extracted by the method of Bligh and Dyer (7). The following day, aliquots of the chloroform layer of each tube were transferred to polyethylene counting vials, the solvent was evaporated under air, and Optiphase HiSafe II scintillant was added prior to the determination of radioactivity as previously described (5). The levels of both 3H and 14C were measured and the results were expressed in disintegrations per minute (dpm) after correction for the recovery of 14C and the volume of media extracted. The amount of [3H]phospholipids secreted by cells was then calculated as follows:

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Bio-Gel P-60 chromatography. An aliquot (2.5 ml) of LFS, dissolved in 20 mM NH4HCO3, pH 7.1, was applied to a Bio-Gel P-60 column (98 ⫻ 2.5 cm) equilibrated with the same buffer. Eighty fractions were collected at a flow rate of 2 ml/h. Fractions from each of the above chromatographic separations were tested for protein content (280 nm absorbance) and for salt concentration (except for the Bio-Gel P-60 fractions), prior to being lyophilized. The HiTrap Heparin-Sepharose and DEAE-Sepharose fractions were resuspended in 1 ml 0.02 M NH4HCO3, pH 7.5, desalted by use of a PD-10 Sephadex G-25 column, and again lyophilized. All fractions were then resuspended in BSS and applied to type II pneumocyte cultures for 3 h to test for their effect on deacylation of the secreted PC. In vitro assay for serum PLA2-like activity. The in vitro measurement of PLA2-like activity was based on the method described by Petrovic et al. (51). 1,2-Bisheptanoylthio-glycerophosphocholine was dissolved in ethanol (25 mg/ml) and aliquots (100 ␮l) were dried under N2 in glass screw-cap tubes, which were then stored at ⫺20°C. Immediately before assay, the substrate was resuspended in 1.9 ml of assay buffer (150 mM KCl, 10 mM CaCl2, 50 mM Tris·HCl, pH 7.5) prior to the addition of 0.4 ml of 1 mM DTNB. The resulting mixture was vortexed vigorously for 1 min and used as a substrate solution (190 ␮l) in the enzyme assay (final substrate concentration 2 mM).

RESULTS

Effect of human serum and its components on surfactant phospholipid secretion. The secretion of surfactant phospholipids from cultured fetal type II pneumocytes into the media is stimulated 8.7-fold when these cells are exposed to human serum (Fig. 1A). When the serum is separated into its major components by use of iodixanol, which generates a gradient upon centrifugation and has a very low toxicity in biological systems, the data presented in Fig. 1B demonstrate that the plasma proteins, other than the lipoproteins, are the major component stimulating secretion (P ⬍ 0.001). HDLs also stimulate phospholipid secretion (P ⬍ 0.002), but with a lesser effect than the plasma proteins, whereas LDLs and VLDLs had no significant effect on the extent of secretion. Given this marked effect of the plasma protein fraction on the rate of secretion of phospholipids, commercially available LFS was examined and also shown to significantly enhance phospholipid secretion (Fig. 2). This effect is evident as early as 30 min after its addition (P ⬍ 0.001) and reached a peak after 3 h of exposure (Fig. 2B). It is also concentration dependent, with a stimulatory effect apparent at a concentration as low as 0.5% (P ⬍ 0.02), and appears to be close to maximal at 4.0%, at which concentration it induced a 2.6-fold increase (Fig. 2A).

Fig. 2. Effect of lipoprotein-free serum (LFS) on phospholipid secretion by cultured fetal type II pneumocytes. Secretion of cellular phospholipids from type II cells was measured 3 h after the addition of the indicated concentrations of LFS (A) or at the indicated times after the addition of either 4% LFS () or vehicle (Œ) (B). Data represent means ⫾ SE of 4 (A) or 3 (B) separate experiments.

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Fig. 1. Effect of human serum (HS) and serum-derived fractions on the secretion of phospholipids from type II pneumocytes. A: secretion of surfactant phospholipids from cultured fetal type II cells was measured after the addition of either 4% human serum or an equivalent volume of water (control plates). B: phospholipid secretion was determined after the addition of vehicle (control), plasma proteins (PP), or the indicated human serum lipoprotein fractions at an equivalent concentration to that of 4% serum. Values represent means ⫾ SE for 3 independent experiments.

After the addition of 10 ␮l of the sample being assayed, the mixtures were incubated in microtiter plates at room temperature for 2 h and the absorbance was determined by using a Bio-Rad 550 microplate reader at both 415 and 595 nm (Bio-Rad Laboratories, Hercules, CA). Expression of results and statistical analysis. The level of secretion was expressed as the percentage of radiolabeled cellular phospholipids that were secreted into the medium during a 3-h incubation. The composition of the secreted phospholipids was ascertained by determining the % of the [3H]choline-containing phospholipids that were lyso-PC, sphingomyelin, or PC. Although variations between replicate cultures of the same batch of cells were always small, the rate of secretion sometimes varied quite markedly between different batches of cells. Subsequently, this was shown to be due to variations in the cell density of the type II cell cultures at the time of measuring the rate of secretion (data not shown). Thus statistical analyses were performed by the paired Student’s t-test except for the in vitro measurement of PLA2-like activity, which was analyzed by multivariate analysis of variance.

ALBUMIN-ASSOCIATED PLA2 ACTIVITY INACTIVATES SURFACTANT PC

Effect of human serum and its components on the composition of secreted phospholipids. Thin-layer chromatographic analysis of the secreted, [3H]choline-containing phospholipids (Fig. 3A) revealed that exposure of the cells to LFS did not effect the sphingomyelin content (P ⬎ 0.05) but led to a significant elevation in the lyso-PC level (5.4-fold; P ⬍ 0.001) and a corresponding decline in the PC level (0.77-fold; P ⬍ 0.001). In contrast, when type II pneumocytes were incubated in the presence of LFS, the cellular phospholipid composition showed no significant change (P ⬎ 0.5; Fig. 3B). Moreover, the level of lyso-PC within the type II cells was very low (⬍1.0%) in both the absence and presence of LFS. These data suggest that a component of serum, most likely a PLA2, is responsible for deacylation of the PC, thereby converting it to lyso-PC, but only after it has been secreted from the cells. When the effect of whole serum (human) and its various components on the composition of the secreted phospholipids was examined (Fig. 4) it was observed that LFS significantly

Fig. 4. Effect of human serum, LFS, and serum lipoproteins on the lyso-PC content of the phospholipids secreted by cultured fetal type II pneumocytes. Confluent cultures of fetal type II cells were exposed to either HS (A), LFS (B), or the indicated lipoproteins at concentrations equivalent to that of 4% serum. After 3 h, the phospholipids secreted from the cells were extracted and the lyso-PC content was determined. Results represent means ⫾ SE of 3 separate experiments. AJP-Lung Cell Mol Physiol • VOL

Fig. 5. Effect of LFS concentration on the lyso-PC content of the phospholipids secreted by cultured fetal type II pneumocytes. After exposure of prelabeled fetal type II cells to the indicated concentrations of LFS for 3 h, the lyso-PC content of the media phospholipids was determined. Results represent means ⫾ SE of 3 separate experiments.

increased the lyso-PC levels in media phospholipids (P ⬍ 0.001) to an extent that was only marginally less than that induced by whole serum. There were also smaller, but significant, elevations in the level of media lyso-PC when the cells were exposed to HDL and LDL [2.6-fold (P ⬍ 0.01) and 1.7-fold (P ⬍ 0.02), respectively]. The extent to which LFS enhanced the lyso-PC content of the secreted lipids was shown to be concentration dependent with a significant elevation being evident at an LFS concentration of 0.5% (P ⬍ 0.001) and the effect being near maximal at a concentration of 4% (Fig. 5). Chromatographic and electrophoretic separation of the serum PLA2-like activity. Given that secretory PLA2, specifically its carboxy-terminal region, has a high affinity for heparin (29, 35), a HiTrap Heparin-Sepharose column was used to characterize the LFS component responsible for the conversion of PC to lyso-PC. The results show that, whereas the secretory form of PLA2 bound to the column and required salt to elute it (Fig. 6C), the PLA2-like activity in LFS had quite different chromatographic properties and did not bind to the column (Fig. 6A). Because ion-exchange chromatography has been previously used to purify PLA2 (40), the chromatographic elution of both the LFS component and the secretory form of PLA2 from a DEAE-Sepharose CL-6B column was examined. Whereas the enzyme activity of LFS is adsorbed to the column and eluted at a KCl concentration of 0.18 – 0.20 M (Fig. 6B), the secretory form of PLA2 is eluted at a much lower KCl concentration (0.08 – 0.13 M; Fig. 6D). The combined data thus demonstrate that the PLA2-like activity of LFS is biochemically distinct from the secretory form of PLA2. When the serum enzyme responsible for inactivation of surfactant phospholipids was subject to chromatography on a Bio-Gel P-60 column (Fig. 7), its activity, evident from the large increase in lyso-PC and associated decrease in PC, eluted in the same fractions as albumin. In addition, conventional analytical electrophoresis was used to separate and elute the LFS components. During electroelution, the serum proteins were mobilized from the gel and eluted into an electrode solution by using a reverse electric field to achieve a high

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Fig. 3. Comparison of the media and cellular phospholipids of cultured type II pneumocytes incubated in the absence or presence of LFS. Three hours after the addition of either 4% LFS (hatched bars) or vehicle (open bars) to cultured type II cells, previously labeled with [3H-methyl]-choline, the radiolabeled phospholipids were extracted from the media and cells and subjected to TLC to separate and measure the relative proportion of lyso-phosphatidylcholine (lyso-PC), sphingomyelin, and phosphatidylcholine (PC). Data represent means ⫾ SE for 3 separate experiments.

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Fig. 6. Elution profile of the phospholipase A2 (PLA2)-like activity of LFS and secretory PLA2 subjected to Heparin- and DEAE-Sepharose chromatography. LFS or secretory PLA2 (sPLA2) was applied to a HiTrap Heparin-Sepharose column (A, C) or a DEAESepharose column (B, D) and eluted as described in MATERIALS AND METHODS. For each fraction, the absorbance at 280 nm (Abs 280) and the salt concentration ([Salt]) were measured prior to determining the extent of hydrolysis of PC secreted from type II pneumocytes. The data depict the lyso-PC content expressed as a percentage of the total radiolabeled lipids in the media.

Fig. 7. Gel filtration chromatography of the PLA2-like activity of LFS. LFS was applied to a Bio-Gel P-60 column and eluted as described in MATERIALS AND METHODS. After lyophilization and resuspension in balanced salts solution, the fractions were assayed for their effect on the proportion of the phospholipid secreted from type II cells that is PC (Œ), sphingomyelin (□), and lyso-PC (). Arrows indicate the position of elution of bovine serum albumin (1), ovalbumin (2), and carbonic anhydrase (3). AJP-Lung Cell Mol Physiol • VOL

activity was also apparent when the enzymatic activity was assessed via an in vitro assay (Fig. 8B). Competitive inhibitors of the different forms of PLA2 were tested in an attempt to characterize the form of the enzyme present in LFS. In particular, p-bromophenacyl bromide (41) and aristolochic acid (64) were used as specific inhibitors of the secretory form of PLA2, whereas palmitoyl trifluoromethyl ketone (PACOCF3) (41) was used as a competitive inhibitor of some Ca2⫹-independent forms of PLA2. None of these inhibitors had any effect upon the serum-induced PC hydrolysis when tested both in culture and in vitro (data not shown). When type II pneumocytes were incubated with HSA V there was a significant change in the lyso-PC content of the media phospholipids (Fig. 9A). Similar results were obtained with crystalline and globulin-free/fatty acid-free commercial preparations of human serum albumin (data not shown). Given that commercially available, serum-derived albumin contains other serum components, either bound to or comigrating with albumin, it is not possible to ascribe the effect specifically to albumin. However, rHSA, which is devoid of these other components, was shown to be equally effective in stimulating the production of lyso-PC in the media (Fig. 9A) (5.3-fold increase compared with control, P ⬍ 0.005). This suggests that the PLA2-like activity that is principally responsible for the production of lyso-PC is, in fact, an inherent activity of albumin. The in vitro effect of LFS and rHSA on lysophospholipid generation from the artificial substrate, thioglycerophospholipid (Fig. 9B), was shown to be comparable to their ability to enhance lyso-PC generation in the media of cultured pneumocytes. As is evident from Fig. 9B, rHSA was shown to be almost as efficient as LFS in stimulating the production of the free thiol (5.7-fold increase compared with control, P ⬍ 0.001). Further evidence that albumin directly converts PC to lyso-PC was obtained when radiolabeled surfactant lipids secreted from cultured fetal type II cells were incubated with rHSA, in vitro in the absence of cells. The data presented in Table 1 show that, upon incubation of the labeled PC with rHSA there was a significant increase in the lyso-PC content (2.2-fold increase compared with control, P ⬍ 0.01) and a concomitant decrease in the level of PC. Moreover, given that

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degree of recovery (92.5%, data not presented). Analysis of the 30 isolated fractions revealed that the factor responsible for the hydrolysis of PC eluted as a single peak, which corresponded with the major protein peak, i.e., serum albumin (data not shown). Characterization of the serum PLA2-like activity of serum. Various concentrations of the Ca2⫹-chelating agent EGTA were evaluated for their effect on the LFS-induced generation of lyso-PC in the media. As shown in Fig. 8A, the inhibitory effect of EGTA is concentration dependent and the serum PLA2-like activity was largely eliminated at 5 mM EGTA (P ⬍ 0.01). These findings demonstrate that the serum enzyme, which stimulates the hydrolysis of PC in secreted surfactant, is Ca2⫹ dependent. Such a conclusion was confirmed when a similar inhibitory effect of EGTA on the serum PLA2-like

ALBUMIN-ASSOCIATED PLA2 ACTIVITY INACTIVATES SURFACTANT PC

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Fig. 8. Inhibitory effect of EGTA on the serum-enhanced generation of lyso-PC. A: fetal type II pneumocytes were exposed to the indicated concentrations of EGTA in the presence of either vehicle or LFS (4% final concentration). The media phospholipids were then extracted and the lyso-PC content was determined. B: 1,2 bisheptanoylthio-glycerophosphocholine (1,2-bisHGPC) was dissolved in assay buffer containing the indicated concentrations of EGTA and, after the addition of LFS, was incubated for 2 h. The rate of generation of lyso-phospholipid was ascertained as described in MATERIALS AND METHODS. Data represent means ⫾ SE of 3 separate experiments.

DISCUSSION

The addition of serum to the culture medium has been shown to enhance the rate of proliferation of fetal type II cells (55) and, in adult cells, stimulate surfactant PC synthesis, preserve lamella-body ultrastructure, and promote multivesicular body production (11). In the present study, serum has been also shown to markedly stimulate the rate of surfactant phospholipid secretion from cultured fetal rat type II pneumocytes. Although HDL stimulated phospholipid secretion, as previously reported by Voyno-Yasenetskaya et al. (65), our studies have shown that it is the plasma proteins, other than the lipoproteins, that are principally responsible for this elevated rate of surfactant lipid secretion. Therefore, in all subsequent experiments we used LFS, which was demonstrated to enhance the rate of phospholipid secretion in a concentration- and time-dependent manner (Fig. 2). In marked contrast to these positive effects, various serum components, in particular a number of serum proteins, have been shown to cause severe inhibition of both endogenous and exogenous surfactant function (10, 17, 37, 56). Physical competition between serum proteins and surfactant phospholipids at the air-liquid interface during the process of adsorption has been shown to lead to surfactant being less effective in low-

ering surface tension (26, 63, 69). Chemical interactions between serum proteins and surfactant phospholipids can result in alterations to the surfactant components (2, 68). In the present study, exposure of type II cells to serum causes a marked change in the composition of the choline-containing phospholipids, which is likely to be the result of a conversion of PC to lyso-PC. However, this is only evident in the phospholipids that have been secreted, not in those within the type II cells (Fig. 3), indicating that this chemical change is only apparent once the lipids have been secreted from the cells. When the effects of the various components of serum were examined, it is clear that, whereas HDL and LDL significantly increase the generation of lyso-PC, the major contributor to the hydrolysis of PC is a component of serum other than the lipoproteins (Fig. 4). Thus the addition of LFS to type II cells results in a concentration-dependent increase in the lyso-PC content of the secreted phospholipids that is almost as great as that produced by whole serum. Given that both in vitro and in vivo studies have shown that elevated levels of lyso-PC can lead to impaired surfactant function (9, 22) and contribute to injurious effects on the alveolar epithelium (48), serum-induced lyso-PC production needs to be investigated as a possible contributor to the development of lung diseases such as ARDS. Seidner et al. (57) have shown that intratracheally administered lyso-PC is rapidly taken up by the lung and intracellularly converted to PC. Our observation that type II cells, when

Fig. 9. Effect of LFS, human serum albumin fraction V (HSA V), and recombinant human serum albumin (rHSA) on lyso-PC production. C, control. A: fetal type II pneumocytes were exposed to vehicle, LFS, HSA fraction V or rHSA prior to determining the lyso-PC content of the secreted phospholipids. B: 1,2-bisHGPC dissolved in assay buffer was incubated with either LFS or rHSA (50 mg/ml) for 2 h. The PLA2-like activity results in an elevation of the free thiol on the lyso-phospholipid, which was measured at 415 nm. Data represent means ⫾ SE of 3 separate experiments.

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the rHSA used in Fig. 9 was no longer commercially available, the data presented in Table 1 were obtained by using a different source of rHSA.

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Table 1. In vitro effect of exposure to LFS and rHSA on lyso-PC content of media phospholipids previously secreted from type II pneumocytes LysoPC Sphingomyelin PC

Control

LFS

rHSA

7.7 ⫾ 1.6 8.8 ⫾ 1.4 83.5 ⫾ 2.8

16.5 ⫾ 3.6* 7.8 ⫾ 0.9 75.7 ⫾ 4.2†

16.6 ⫾ 3.8* 8.0 ⫾ 1.0 75.3 ⫾ 4.5†

exposed to serum, accumulate lyso-PC in the surrounding media could be explained if a serum component inhibited the uptake and subsequent recycling of lyso-PC. In this context, it is pertinent that serum albumin has been shown to bind directly to lyso-PC (36). Another possibility is that the accumulation of lyso-PC in the media is the result of a direct hydrolysis of surfactant PC to lyso-PC by a serum PLA2-like enzymatic activity. If this is the case, then it needs to be considered that there are three enzymes known to be present in serum that have the potential to bring about this conversion, namely sPLA2 (EC 3.1.1.4) (3), platelet-activating factor acetylhydrolase (PAFAH; EC 3.1.1.47) (61), and lecithin:cholesterol acyltransferase (LCAT; EC 2.3.1.43) (1). In an attempt to identify the serumderived PLA2-like activity, LFS and sPLA2 were subjected to chromatography. The data depicted in Fig. 6, A and C, show that the PLA2-like activity of LFS does not bind to HiTrap Heparin-Sepharose (29), unlike that of sPLA2, which does bind (35). The LFS-derived PLA2-like activity, when subjected to DEAE-Sepharose chromatography (40), also showed different chromatographic characteristics than that of sPLA2 and it was apparent that the serum enzyme activity coeluted with albumin (Fig. 6B). This latter attribute was also evident when LFS was subjected to chromatography on a BioGel P60 column (Fig. 7) or subjected to polyacrylamide electrophoresis (data not shown). Because EGTA inhibits the serum PLA2-like enzyme activity (Fig. 8), it is concluded that the enzyme is calcium dependent, as are the secretory forms of PLA2 from rat lung (40) and human serum (53). Thus, in respect to this attribute, it is similar to members of the sPLA2 family. However, the serum PLA2like activity is not inhibited by either 4-bromo-phenacyl bromide or aristolochic acid, both of which have been reported to inhibit calcium-dependent sPLA2 (42, 64). These findings, together with the above-mentioned chromatographic observations, demonstrate that the serum PLA2-like activity is chemically distinct from sPLA2. Because PAF-AH has been shown to be Ca2⫹ independent (60) and inhibited by PACOCF3 (34), it is apparent that PAF-AH is distinct from the PLA2-like activity of LFS, since the latter is Ca2⫹ dependent and not affected by this inhibitor. Moreover, 70% of human serum AJP-Lung Cell Mol Physiol • VOL

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Results represent means ⫾ SE of 4 separate experiments. Lung type II pneumocytes were isolated, cultured, and labeled as described in Fig. 2. The cultured type II cells were then thoroughly washed with balanced salts solution and equilibrated for 3 h at 37°C in serum-free MEM. The media was then collected and centrifuged at 300 g for 6 min to remove any detached cells, and 1.7-ml aliquots of the supernatant containing the secreted radiolabeled phospholipids were further incubated for 3 h in the presence of 70 ␮l of either lipoprotein-free serum (LFS) or recombinant human serum albumin (rHSA) (final concentrations 4% and 2 mg/ml, respectively). An equivalent volume of vehicle (water) was added to control plates. The percentages of lyso-phosphatidylcholine (lyso-PC), sphingomyelin, and phosphatidylcholine (PC) in the secreted lipids were then determined. *Significantly different (P ⬍ 0.01) from that of controls. †Significantly different (P ⬍ 0.05) from that of controls.

PAF-AH is associated with LDL and 30% bound with HDL (61) and thus would not be present in LFS. The fact that the PLA2-like activity of LFS coelutes with standard albumin (Fig. 7) suggests that the enzymatic activity has a molecular ratio (Mr) of 67,000, which also corresponds to the estimated Mr of LCAT (66 – 68,000) (1). However, LCAT is found predominantly associated with HDL (46), and its activity is destroyed if heat treated at 58°C for 20 min (1, 32), neither of which are properties of the PLA2-like activity of LFS. Note that the PLA2-like activity detected after chromatography on Heparin- and DEAE-Sepharose columns was highly active despite the fact that the LFS had been subjected to heat treatment at 60°C for 30 min prior to chromatography (see Fig. 6). The present study has demonstrated that HSA V leads to an elevated content of lyso-PC in the media phospholipids, which is of the same magnitude as that generated by LFS (Fig. 9). Thus it is possible that HSA has an associated PLA2-like activity, which is in agreement with a previous report that there is an association between serum PLA2 activity and several commercially available albumin preparations (14). A study by Langton and Dench (38) confirmed this association by demonstrating that, whereas a single zone of PLA2 activity was detected when purified human PLA2 was subjected to agarose electrophoresis, an additional zone of activity, corresponding to the position of migration of the albumin, was observed when the purified enzyme was mixed with albumin (HSA V) prior to electrophoresis. We hypothesized that rHSA would be devoid of PLA2-like activity because it is produced by using a mammalian cell line grown under conditions devoid of any humanor animal-derived serum and is therefore not likely to be bound with PLA2. However, the present study has revealed that exposure of type II cells to rHSA is almost as effective as LFS in enhancing the level of lyso-PC in the media (Fig. 9A). This implies that rHSA is able to directly generate lyso-PC and suggests that the PLA2 may be an innate activity of albumin. Such a proposal is supported by the observations that rHSA is also able to hydrolyze a synthetic phospholipid in an in vitro assay (Fig. 9B) and that when rHSA is incubated in vitro with secreted radiolabeled surfactant lipids there is a resultant increase in lyso-PC (Table 1). In the latter case, the extent of lyso-PC generation is less than that observed when the rHSA is incubated directly with prelabeled type II cells. This lower response may be due to an interaction between the albumin and the cells or it might be due to differences in the susceptibility to albumin-induced hydrolysis of the different physical forms (e.g., tubular myelin, surfactant lipid monolayers, etc.) of the secreted surfactant lipids. Various lung injuries lead to leakage of serum components into the alveoli and these have been shown to have a significant adverse effect on pulmonary surfactant function often leading, in the case of adults, to the development of ARDS. Attempts to overcome the adverse outcomes of ARDS by using surfactant replacement therapy have proven to be relatively unsuccessful (39, 44, 59), suggesting that under these pathological conditions the surfactant, including any exogenous surfactant introduced for therapeutic purposes, is rendered inactive. Numerous studies have focused on the capacity of serum components to induce surfactant dysfunction (26, 37, 56). In particular, surface-active serum proteins, such as albumin, have been shown in numerous studies to impair the adsorption of lung surfactant

ALBUMIN-ASSOCIATED PLA2 ACTIVITY INACTIVATES SURFACTANT PC

ACKNOWLEDGMENTS The generous financial support provided through the award of an Alan Villiers and Iris May Peacocke Scholarship to J. E. Damas is gratefully acknowledged. DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the author(s). REFERENCES 1. Albers JJ, Chen CH, Lacko AG. Isolation, characterization, and assay of lecithin-cholesterol acyltransferase. Methods Enzymol 129: 763–783, 1986. 2. Amirkhanian JD, Bruni R, Waring AJ, Taeusch HW. Inhibition of mixtures of surfactant lipids and synthetic sequences of surfactant proteins SP-B and SP-C. Biochim Biophys Acta 1096: 355–360, 1991. AJP-Lung Cell Mol Physiol • VOL

3. Arbibe L, Koumanov K, Vial D, Rougeot C, Faure G, Havet N, Longacre S, Vargaftig BB, Brerzist G, Voelker DR, Wolf C, Touqui L. Generation of lyso-phospholipids from surfactant in acute lung injury is mediated by type-II phospholipase A2 and inhibited by a direct surfactant protein A-phospholipase A2 protein interaction. J Clin Invest 102: 1152– 1160, 1998. 4. Arbibe L, Vial D, Touqui L. Phospholipase A2 and acute respiratory distress syndrome. Prog Surg 24: 79 –87, 1997. 5. Asokananthan N, Cake MH. Stimulation of surfactant lipid secretion from fetal type II pneumocytes by gastrin-releasing peptide. Am J Physiol Lung Cell Mol Physiol 270: L331–L337, 1996. 6. Attalah HL, Wu Y, Alaoui-El-Azher M, Thouron F, Kumaniv K, Wolf C, Brochard L, Harf A, Delclaux C, Touqui L. Induction of type-IIA secretory phospholipase A2 in animal models of acute lung injury. Eur Respir J 21: 1040 –1045, 2003. 7. Bligh EG, Dyer WJ. A rapid method of total lipid extraction and purification. Can J Biochem Physiol 37: 911–917, 1959. 8. Chotigeat U, Promwong N, Kanjanapattanakul W, Khorana M, Sangtawesin V, Horpaopan S. Comparison outcome of surfactant therapy in respiratory distress syndrome in two periods. J Med Assoc Thai 3: 109 –114, 2008. 9. Cockshutt AM, Possmayer F. Lysophosphatidylcholine sensitizes lipid extracts of pulmonary surfactant to inhibition by serum proteins. Biochim Biophys Acta 1086: 63–71, 1991. 10. Cockshutt AM, Weitz J, Possmayer F. Pulmonary surfactant associated protein A enhances the surface activity of lipid extract surfactant and reverses inhibition by blood protein in vitro. Biochemistry 29: 8424 –8429, 1990. 11. Cott GR, Walker SR, Mason RJ. The effect of substratum and serum on lipid synthesis and morphology of alveolar type II cells in vitro. Exp Lung Res 13: 427–447, 1987. 12. Dobbs LG, Gonzalez R, Williams MC. An improved method for isolating type II cells in high yield and purity. Am Rev Respir Dis 134: 141–145, 1986. 13. Edelson JD, Vadas P, Villar J, Mullen JBM, Pruzanski W. Acute lung injury induced by phospholipase A2. Structural and functional changes. Am Rev Respir Dis 142: 1102–1109, 1991. 14. Elsbach P, Pettis P. Phospholipase activity associated with serum albumin. Biochim Biophys Acta 296: 89 –93, 1973. 15. Fernsler JG, Zasadzinski JA. Competitive adsorption: a physical model for lung surfactant inactivation. Langmuir 25: 8131–8143, 2009. 16. Finkelstein JN, Maniscalco WM, Shapiro D. Properties of freshly isolated type II epithelial cells. Biochim Biophys Acta 762: 398 –404, 1983. 17. Fuchimukai T, Fujiwara T, Takahashi A, Enhorning G. Artificial pulmonary surfactant inhibited by proteins. J Appl Physiol 62: 429 –437, 1987. 18. Galani V, Tatsaki E, Bai M, Kitsoulis P, Lekka M, Nakos G, Kanavaros P. The role of apoptosis in the pathophysiology of Acute Respiratory Distress Syndrome (ARDS): an up-to-date cell-specific review. Pathol Res Pract 206: 145–150, 2010. 19. Goerke J. Pulmonary surfactant: functions and molecular composition. Biochim Biophys Acta 1408: 79 –89, 1998. 20. Griese M. Pulmonary surfactant in health and human lung diseases: state of the art. Eur Respir J 13: 1455–1476, 1999. 21. Gross L, Wilson CM, Ingleson LD, Brehier A, Rooney SA. The influence of hormones on the biochemical development of fetal rat lung in organ culture: I. Estrogen. Biochim Biophys Acta 575: 375–383, 1979. 22. Grossmann G, Tashiro K, Kobayashi T, Suzuki Y, Matsumoto Y, Waseda Y, Akino T, Curstedt T, Robertson B. Experimental neonatal respiratory failure induced by lysophosphatidylcholine: effect of surfactant treatment. J Appl Physiol 86: 633–640, 1999. 23. Gunther AD, Valmrath R, Grimminger F, Seeger W. Alteration of pulmonary surfactant in ARDS-pathogenetic role and therapeutic perspectives. In: Acute Respiratory Distress Syndrome: Cellular and Molecular Mechanisms and Clinical Management. New York: Plenum, 1998, p. 97–106. 24. Hallman M, Spragg R, Harrell JH, Moser KM, Gluck L. Evidence of bronchoalveolar lavage phospholipids, surface activity, and plasma myoinositol. J Clin Invest 70: 673–683, 1982. 25. Hite RD, Seeds MC, Safta AM, Jacinto RB, Gyves JI, Bass DA, Waite BM. Lysophospholipid generation and phosphatidylglycerol depletion in phospholipase A2-mediated surfactant dysfunction. Am J Physiol Lung Cell Mol Physiol 288: L618 –L624, 2005.

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from the subphase to the air-liquid interface within the alveoli (15, 54, 69, 71). The present study, however, has demonstrated that albumin is also able to convert PC, the major surfaceactive component of surfactant, to lyso-PC via an intrinsic PLA2-like activity. Elevated levels of lyso-PC have been shown to lead to surfactant inactivation in both in vivo (22, 48) and in vitro studies (9, 25). Thus any albumin accumulating in the alveolar fluid as a consequence of transient lung injury could result in the development of ARDS as a direct consequence of its associated PLA2-like activity. Recognition that albumin has an associated PLA2 activity could also provide a rationale for the development of novel treatment regimes designed to minimize lung injury in patients showing symptoms of ARDS and a means of protecting infused synthetic surfactant lipids from rapid degradation. Such an outcome would be more likely if a specific, nontoxic inhibitor could be identified that would minimize the damaging effects of the albumin-associated PLA2. Previous attempts to improve the efficacy of therapies used in the treatment of ARDS patients have focused on the use of various additives, such as chitosan and hyaluronan (HA), which have been shown to be effective in both in vitro and animal models (43, 70). One such study has shown that the inhibition of pulmonary surfactant induced by exposure to PLA2 is attenuated by HA through its ability to diminish the PLA2 activity (31). Based on the anti-inflammatory effects of HA and its ability to reduce PLA2 activity, and thus minimize surfactant inhibition, it has been suggested that concomitant treatment with HA and surfactant might provide a promising approach to the treatment of ARDS. An alternative approach, described in recent papers, has focused on the use of PLA2-resistant surface-active phosphonolipids to develop new artificial surfactants (49, 66, 67), which may eventually prove to have advantages in the treatment of ARDS. In summary, although inhibition of pulmonary surfactant by albumin has been the subject of numerous studies and albumin has been shown to competitively interfere with the generation of a phospholipid film at the air-liquid interface (54, 69, 71), the mechanism(s) by which this protein inactivates surfactant lipids remains controversial. Our observation that serum contains an albumin-associated PLA2-like activity, which leads to the accumulation of lyso-PC in secreted surfactant phospholipids, may be another attribute of albumin that contributes to reduced efficacy of exogenous surfactants in the treatment of ARDS.

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