Localization of a candidate surfactant convertase to type II cells, macrophages, and surfactant subfractions

Howard Clark, Lennell Allen, Erin Collins, Frederick Barr, Leland Dobbs, Gunther Putz, Jon Goerke, and Samuel Hawgood

Cardiovascular Research Institute and Department of Pediatrics, University of California, San Francisco, California 94118-1245


    ABSTRACT
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Abstract
Introduction
METHODS
RESULTS
DISCUSSION
References

Pulmonary surfactant exists in the alveolus in several distinct subtypes that differ in their morphology, composition, and surface activity. Experiments by others have implicated a serine hydrolase in the production of the inactive small vesicular subtype of surfactant (N. J. Gross and R. M. Schultz. Biochim. Biophys. Acta 1044: 222-230, 1990). Our laboratory recently identified this enzyme in the rat as the serine carboxylesterase ES-2 [F. Barr, H. Clark, and S. Hawgood. Am. J. Physiol. 274 (Lung Cell. Mol. Physiol. 18): L404-L410, 1998]. In the present study, we determined the cellular sites of expression of ES-2 in rat lung using a digoxygenin-labeled ES-2 riboprobe. ES-2 mRNA was localized to type II cells and alveolar macrophages but not to Clara cells. Using a specific ES-2 antibody, we determined the protein distribution of ES-2 in the lung by immunohistochemistry, and it was found to be consistent with the sites of mRNA expression. Most of the ES-2 in rat bronchoalveolar lavage is in the surfactant-depleted supernatant, but ES-2 was also consistently localized to the small vesicular surfactant subfraction presumed to form as a consequence of conversion activity. These results are consistent with a role for endogenous lung ES-2 in surfactant metabolism.

carboxylesterase; ES-2; diisopropyl fluorophosphate-binding protein


    INTRODUCTION
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Abstract
Introduction
METHODS
RESULTS
DISCUSSION
References

A THIN LAYER OF FLUID covers the extensive luminal surface of the pulmonary alveolar epithelium. Pulmonary surfactant secreted into this fluid from alveolar type II cells forms a lipoprotein film at the air-fluid interface. By reducing the surface tension of this interface, surfactant contributes to the overall volume stability of the lung throughout respiration (3). The details of how the surface film is maintained during the cyclic expansion and contraction of the respiratory cycle are not fully understood. It is generally believed that specific surfactant apoproteins play a central role in a complex extracellular cycle that both replenishes and clears components of the surface film (26). The cycle starts with secreted lamellar body contents converting to an aggregated lattice of protein-rich membranes known as tubular myelin. The phospholipids making up the surface film are probably adsorbed directly from disassociating tubular myelin. A variety of protein-poor vesicular forms of surfactant are generated during respiration, either as by-products of the structural conversions leading up to the formation of the surface film or as a consequence of film overcompression or desorption during expiration. These vesicles and perhaps other surfactant fractions are efficiently cleared from the alveolar space by type II cells and alveolar macrophages (27).

The composition and biophysical properties of each surfactant subtype have been described with traditional biochemical and surface balance techniques (14, 17). The factors controlling the dynamic conversion of one surfactant subtype to the other during repeated cycles of expansion and compression have only recently come under study. Despite the apparent importance of this phenomenon in normal surfactant homeostasis, only a single model system has been developed to study the regulation of subtype conversion in vitro. In the simple rotating-tube model of the cycling interface introduced by Gross and Narine (7), fractions of surfactant rich in tubular myelin can be reproducibly converted to apoprotein-poor vesicles of a similar composition, density, morphology, and activity to those isolated from lung lavage. During a search for factors that might regulate this conversion, Gross and Schultz (8) established that serine hydrolase inhibitors block the conversion of freshly secreted mouse surfactant to light vesicles. This observation suggested that a serine hydrolase played a role in the surfactant subtype conversion associated with a cyclically compressed interface. Although the specificity of this effect has been challenged (10), Gross (6) has gone on to implicate a specific serine carboxylesterase present in mouse bronchoalveolar lavage (BAL) fluid in the regulation of surfactant. In both rat and mouse, this enzyme has recently been identified as a serine carboxylesterase (1, 13). Importantly, a partially purified preparation of the mouse esterase promotes the conversion of both mouse surfactant and reconstituted surfactant (4, 13).

Minimal requirements for a specific role for this carboxylesterase in surfactant subtype conversion in vivo and in vitro are that it should be present in the alveolar space and associated with fractions active in the cycling assay. To determine whether these criteria are met, we have characterized the distribution of protein and mRNA in cells of the distal rat lung and the distribution of ES-2 in subfractions of rat BAL fluid. By corollary, surfactant lacking the enzyme should not cycle to a less-dense inactive form. This was tested in the present study by cycling surface-active reconstituted tubular myelin lacking ES-2. Our results support a role for ES-2 in extracellular surfactant subtype conversion, but definitive proof of such a role must wait until the ability to purify or express sufficient enzyme is achieved to test its activity directly.


    METHODS
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Abstract
Introduction
METHODS
RESULTS
DISCUSSION
References

Antibody production. Several intracellular esterases are expressed in the lung. The protein sequences of the five cloned rat carboxylesterases share up to 96% identity (30), and polyclonal antibodies directed against full-length esterases can show considerable cross-reactivity (28, 29). We therefore adopted a synthetic peptide strategy previously shown (16) to clearly distinguish ES-2 from homologous intracellular esterases. A peptide with the sequence CNPPQTEHTEHT, corresponding to the last 11 amino acids of ES-2, and an NH2 cysteine for purposes of conjugation was synthesized by standard techniques. This peptide was conjugated to keyhole limpet hemocyanin and used to raise a polyclonal antibody in rabbits with standard immunization protocols. Specific anti-ES-2 IgG was isolated from the immune serum by peptide-affinity chromatography. Our laboratory previously reported (1) that this antibody recognizes a single protein of 68 kDa in both rat serum and BAL fluid, consistent with the predicted size of glycosylated ES-2.

Polyclonal antibodies against sheep surfactant protein (SP) B and sheep SP-A were raised in rabbits with standard techniques as previously described (12). The monoclonal antibody directed against a rat type II cell surface antigen has also been previously characterized (5).

Immunohistochemistry. The lungs of adult Sprague-Dawley rats were initially fixed by in situ tracheal instillation of 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4. After removal, they were immersed in the same fixative at room temperature for 60-90 min. The lungs were then cut into pieces and fixed further in fresh 4% paraformaldehyde in 0.1 M phosphate buffer at 4°C for 3 h and then overnight at 4°C in 4% paraformaldehyde in 0.1 M phosphate buffer with 30% sucrose. Tissue samples were then embedded in Tissue-Tek optimum cutting temperature compound (Miles, Elkhart, IN), frozen in Freon-22, and stored in liquid nitrogen.

Cryostat sections (3 µm) mounted onto Fisher Superfrost Plus slides and stored at -20°C were thawed and washed in a buffer of PBS containing 0.1% BSA and 0.3% Triton X-100. The sections were incubated with this buffer plus 10% goat serum for 1 h at room temperature to block nonspecific binding. The tissue sections were then incubated with specific anti-ES-2 IgG at 4°C overnight. After being washed with PBS containing 0.1% BSA and 0.3% Triton X-100, sections were treated with fluorescein-coupled goat anti-rabbit IgG secondary antibody (Cappel, Durham, NC) for 1 h. Sections were then washed sequentially in PBS followed by double-distilled water. After coverslips were mounted, the bound antibodies were detected under fluorescence microscopy. Control sections treated with secondary antibody alone were negative in all experiments.

In situ hybridization. The 454-bp lung ES-2 cDNA, subcloned into a pCR II vector as previously described (1), were used to generate sense and antisense riboprobes labeled with digoxigenin. The clones were linearized, and riboprobe synthesis was performed with an RNA transcription kit (Boehringer Mannheim, Indianapolis, IN) and RNA polymerase promoters SP-6 and T7. Three-micrometer cryosections of rat lung were thawed to room temperature, dried at 55°C, and then fixed in 4% paraformaldehyde in PBS at room temperature for 30 min. After treatment with proteinase K (10 µg/ml in 20 mM Tris and 1 mM EDTA, pH 7.2) for 5 min at room temperature and further washes in PBS followed by 2× saline-sodium citrate (SSC), hybridization was performed overnight at 65°C with 50 ng digoxigenin-labeled riboprobe/ml hybridization solution containing 40% formamide, 5× SSC, 1× Denhardt's solution, 100 µg/ml of salmon testis DNA, and 100 µg/ml of tRNA. After hybridization, the sections were washed three times for 20 min in 5× SSC and for 40 min at 60°C in 0.5× SSC containing 20% formamide. After a further wash at 37°C in 0.5 M NaCl-10 mM Tris (pH 7.0)-5 mM EDTA for 15 min, sections were digested with RNase A (10 µg/ml) and washed in 0.5 M NaCl-10 mM Tris (pH 7.0)-5 mM EDTA and then in 0.5× SSC containing 20% formamide for 30 min at 60°C. After a final rinse in 2× SSC for 30 min at room temperature, the sections were incubated overnight at 4°C with a 1:5,000 dilution of anti-digoxigenin-alkaline phosphatase conjugate (Boehringer Mannheim) in a moist chamber. Hybridization of the sections with sense-direction riboprobes and development of the slides with secondary antibody only served as negative controls.

Surfactant isolation. Adult Sprague-Dawley rats were injected intraperitoneally with 65 mg of pentobarbital sodium for anesthesia and then exsanguinated via the inferior vena cava. The lungs were perfused with PBS while being repeatedly inflated to total lung capacity with air. BAL was performed with 8 ml of lavage buffer [(in mM) 10 HEPES, 140 NaCl, 2 CaCl2, and 1 MgCl2, pH 7.4] per lavage for a total of eight lavages. The pooled BAL fluid was centrifuged at 150 g for 10 min to remove cells and then at 100,000 g for 180 min in an SW28 rotor to pellet the surfactant. We gently resuspended the pellets in 1 ml of lavage buffer, taking care not to generate foam. The surfactant was further fractionated by centrifugation on a 13-ml sucrose gradient (0.25-0.75 M sucrose in lavage buffer) at 100,000 g for 48 h in an SW41 rotor at 4°C. Fractions of 0.75 ml were collected and analyzed for phosphorus content by the Bartlett (2) assay.

Immunoblot detection of ES-2 in surfactant subfractions. An aliquot from each fraction from the sucrose gradient was analyzed for ES-2 content by standard immunoblot analysis on nitrocellulose after 15% polyacrylamide gel electrophoresis under denaturing conditions in the presence of SDS. Reducing agents were not used to optimize antibody detection of SP-B. The nitrocellulose was blocked with 5% milk, incubated with the ES-2 antiserum at 1:500 dilution, and developed with a goat anti-rabbit IgG secondary antibody and an enhanced chemiluminescence detection system (Amersham). To determine whether the ES-2 present was tightly associated with the particulate surfactant, each fraction was made to 5 ml in lavage buffer to dilute the sucrose and centrifuged at 100,000 g for 60 min. The supernatants were discarded, and the pellets were analyzed by immunoblot.

Reconstitution of surfactant from phospholipids and surfactant apoproteins. Surfactant was reconstituted from dog SP-A, dog SP-B, dipalmitoylphosphatidylcholine, and egg phosphatidylglycerol with the n-octyl glucoside dialysis method described in detail previously (25). Briefly, dipalmitoylphosphatidylcholine, phosphatidylglycerol, and SP-B (7:3:1 wt/wt/wt) were mixed in chloroform-methanol (2:1) and dried as a thin film under vacuum. The lipoprotein film was hydrated in 2 mM Tris-100 mM n-octyl glucoside, pH 7.4, at 40°C for 30 min, diluted fivefold with 10 mM Tris-140 mM NaCl-1 mM EDTA, then dialyzed exhaustively against the dilution buffer at 4°C. After dialysis, SP-A was added (phospholipid-SP-A, 20:1 wt/wt), and the sample was incubated at 37°C for 15 min. CaCl2 was then added to a final calcium concentration of 5 mM, and the incubation was continued for 16 h at 37°C. The sample was then used without storage for cycling, electron microscopy, and surface activity experiments. The density of the reconstituted sample was determined before and after cycling by sucrose gradient centrifugation as described in Surfactant isolation for rat surfactant.

In vitro cycling of surfactant. Rat surfactant or reconstituted surfactant (300 µg of phospholipid in 2 ml of lavage buffer) was added to a 5-ml polystyrene Falcon tube and cycled end over end on a rotating wheel at 40 rpm for 3 h at 4°C. Sham-cycled samples were rotated similarly, except the cyclically expanded and contracted air-liquid interface was avoided with the use of completely liquid-filled 2-ml Eppendorf tubes. Mixing in the sham tubes was achieved by including two glass beads. The samples were tested for surface activity before and after cycling.

Esterase assay. The nonspecific carboxylesterase assay described by Heymann et al. (11) that utilized p-nitrophenyl acetate as a substrate was modified slightly for the measurement of activity in fractions from the sucrose gradient. Briefly, a fresh solution of 0.5 mM p-nitrophenyl acetate in acetone was prepared immediately before use. With the use of temperature-controlled quartz cuvettes in a Gilford response spectrophotometer, the baseline absorbance at 405 nm was established at 37°C, pH 7.4. Aliquots of each fraction of the surfactant sucrose gradient were added at time 0, the cuvette contents were rapidly mixed, and absorbance was recorded every 15 s for 4 min.

Surface activity measurements. The surface activity of rat surfactant and reconstituted surfactant before and after cycling was evaluated in a pressure-driven captive bubble surfactometer with an experimental protocol previously described in detail by our laboratory (19). Samples were tested at a phospholipid concentration of 150 µg/ml in a buffer containing 2 mM CaCl2. After the sample was adsorbed to the bubble surface for 30 min, a slow first-compression surface tension-area isotherm was inscribed by a stepwise increase in system pressure every 60 s from 0.5 to 2.8 atm. After we determined film stability at minimum area for 5 min, the bubble was cycled eight times for 2-3 min between the minimum and maximum areas to determine the minimum surface tension during cycling. A final 10th slow-compression isotherm was then inscribed. The minimum surface tension achieved with each bubble compression was calculated from the bubble dimensions with the method of Malcolm and Elliot (15) as modified by Schürch et al. (21).


    RESULTS
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Abstract
Introduction
METHODS
RESULTS
DISCUSSION
References

Distribution of ES-2 protein and mRNA in the lung. ES-2 was localized to type II cells and alveolar macrophages by immunohistochemistry (Fig. 1). There was also variable weak staining of the alveolar septum, consistent with the presence of ES-2 in the serum. Nonciliated bronchiolar cells or Clara cells in the respiratory bronchioles did not react with the ES-2-specific antiserum. Control slides developed without the primary antiserum were negative (data not shown).


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Fig. 1.   Immunocytochemistry (A and C) and phase contrast (B and D) for ES-2 in rat lung. A and B: adult lung demonstrating cytoplasmic staining of type II (TII) cells. C and D: adult rat lung demonstrating staining of alveolar macrophages (MAC).

The cells expressing ES-2 in the lung were determined by in situ hybridization with a digoxygenin-labeled riboprobe previously shown (1) not to cross-hybridize with ES-10, a homologous and abundant intracellular carboxylesterase expressed in the lung (29). ES-2 mRNA was present in scattered alveolar epithelial cells and alveolar macrophages (Fig. 2A). No ES-2 mRNA was detected in Clara cells (Fig. 2B).


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Fig. 2.   In situ hybridization of ES-2 in rat lung parenchyma. A: sense control. B: antisense showing scattered staining in alveolar walls. C: higher-power view showing scattered staining of alveolar wall cells in distribution of type II cells. D: higher-power view demonstrating staining of TII cell and Mac. Bars show relative magnification between panels.

To confirm that the scattered cells in the alveolar wall positive for ES-2 mRNA were type II cells and not tissue macrophages, colocalization experiments were carried out by probing sections that had undergone the in situ hybridization reactions with an antibody specific for type II cell membrane protein (5). Most cells with a cytoplasmic reaction for ES-2 mRNA also reacted with the type II cell plasma membrane-specific antiserum (Fig. 3). Occasional cells were positive for only one reaction product, presumably because both cytoplasm and plasma membrane were not always present in the 3-µm sections used.


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Fig. 3.   Double labeling of rat lung with ES-2 in situ hybridization and anti-TII cell surface antigen. A: in situ hybridization with digoxigenin-labeled ES-2 probe. B: same section under fluorescence microscopy showing apical staining with anti-TII cell monoclonal antibody.

Distribution of ES-2 and esterase activity in BAL fluid fractions. Semiquantitative dot blots of the 100,000 g-centrifuged lavage supernatant and surfactant pellet showed that ~90% of the BAL ES-2 was present in the lipid-depleted supernatant, whereas ~10% of the total amount of the enzyme in rat BAL fluid was associated with the surfactant pellet. Although centrifugation at 100,000 average g for 3 h may not sediment all of the apoprotein-depleted, less dense forms of surfactant, Barr et al. (1) previously reported that the majority of the ES-2 in the surfactant-depleted supernatant elutes from a sizing column with a predicted molecular mass of 68 kDa, suggesting it is not complexed with lipid or other proteins.

The smaller but consistent amount of ES-2 in the 100,000 g-centrifuged surfactant pellet was associated with fractions with a density of 1.03-1.047 g/ml (Fig. 4; ES-2 peak in fraction 4, density 1.04 g/ml), a density consistent with the light, apoprotein-depleted fraction of rat surfactant. Most of the immunoreactive ES-2 was monomeric after denaturing but nonreducing gel electrophoresis, but faint immunoreactive bands presumably representing ES-2 oligomers were also seen in fractions 5-7. The significance of these oligomers, if any, is not known. The density of the fractions containing ES-2 was slightly greater than the 1.025 g/ml reported for the light subfraction of mouse surfactant. This difference most likely represents interspecies variation rather than heterogeneity in the heavy subfraction of rat surfactant because the less dense fractions did not contain SP-A or SP-B, and the small vesicles produced by rat surfactant with in vitro cycling also had a density of 1.03-1.04 g/ml (data not shown). As expected, the distribution of SP-B (Fig. 4) and SP-A (data not shown) corresponded to the peak of the heavy phospholipid subfractions (Fig. 4; SP-B peak in fraction 7, density 1.057 mg/ml). The same distribution of both ES-2 and SP-B was found by Western analysis of material pelleted from individual fractions at 100,000 g for 1 h, suggesting both proteins were lipid associated. In contrast to the restricted distribution of ES-2, albumin immunoreactivity was detected diffusely across the gradient (data not shown). The restricted distribution of ES-2 suggests an association with specific surfactant subtypes rather than nonspecific lipid association or entrapment.


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Fig. 4.   Identification of ES-2, esterase activity, and surfactant protein (SP) B in rat surfactant subtypes. Rat surfactant purified from bronchoalveolar lavage fluid by centrifugation at 100,000 g for 180 min was fractionated over a sucrose gradient at 100,000 g for 48 h. A: phospholipid content (in µg; ) and esterase activity (in arbitrary units; open circle ) of each fraction. Fractions 1-16 represent top (density 1.01 mg/ml) to bottom (density 1.095 mg/ml) of gradient. B: distribution of ES-2 (68 kDa) and SP-B (18 kDa) across gradient. Nonglycosylated recombinant ES-2 is loaded in lane 0 as a control. *ES-2 oligomers. Profile is representative of 3 experiments.

Although the profile of esterase activity overlapped the peak of ES-2 immunoreactivity, the peak activity was slightly shifted relative to the peak of the immunoreactive ES-2 monomer and appeared to correspond more precisely to the ES-2 oligomer distribution.

In vitro cycling of rat surfactant and reconstituted tubular myelin. Rat surfactant and reconstituted surfactant were cycled in vitro with the protocol of Gross and Narine (7). The main purpose of these experiments was to determine the effect of cycling surfactant preparations on surface activity in the presence and absence of esterase. Before we tested the surface activity, we characterized the density profiles of rat surfactant and reconstituted surfactant before and after in vitro cycling for purposes of comparison with the well-characterized mouse system (7).

A representative phospholipid profile of the control rat surfactant is shown in Fig. 4. The major phospholipid peak before cycling and after sham cycling had a density of 1.057 g/ml, consistent with the heavy subtype described by Gross and Narine (7). Before cycling or after sham cycling, a significant upfield shoulder to the main peak (Fig. 4; fraction 4, density 1.037-1.04 g/ml) corresponding to the light subtype described by Gross and Narine was consistently seen. After cycling, the heavy phospholipid peak shifted to a density of 1.03-1.04 g/ml. The morphology of the peak fractions determined by electron microscopy, before and after cycling were also similar to several previous reports (7, 17), with predominantly tubular myelin and multilamellated forms in the heavy fraction before cycling and predominantly unilamellar vesicles in the light fractions after cycling (data not shown). Also, as previously reported, we did not detect any SP-A or SP-B associated with the peak phospholipid fraction, density 1.03-1.04 g/ml, after cycling. In contrast, the peak density of surfactant reconstituted with a phospholipid-SP-B-SP-A composition of 10:1:1 (wt/wt/wt) was 1.08-1.09 g/ml. The morphology of surfactant reconstituted in this way has been previously described in detail by Williams et al. (25). There was a small shift toward a lighter density of 1.07-1.08 g/ml with cycling. SP-B remained associated with the phospholipid peak after cycling, although there was consistently a small reduction in the SP-A content of the peak fraction (data not shown).

The noncycled and sham-cycled rat surfactants adsorbed rapidly, reaching an equilibrium surface tension of 25 mN/m before beginning the first-compression isotherm. In contrast, the cycled rat surfactant adsorbed slowly, only achieving a surface tension of 50 mN/m by 30 min. The noncycled and sham-cycled samples reached surface tensions of <5 mN/m on the second compression, whereas the cycled sample failed to reduce the surface tension below 25 mN/m (Fig. 5). The results with the control and sham-cycled samples were comparable with previous results with rabbit surfactant (18).


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Fig. 5.   Effect of in vitro cycling on minimum surface tension achieved with repeated captive bubble compression. A: natural rat surfactant. B: reconstituted surfactant. Cycle 0, surface tension at end of adsorption period; cycle 1, surface tension at end of 1st slow isotherm; cycles 2-9, surface tensions at end of rapid compressions; cycle 10, surface tension at end of slow 10th compression. Data are representative of 2 experiments.

In marked contrast to the loss of activity with cycling demonstrated by rat surfactant, reconstituted surfactant had excellent surface activity before and after cycling (Fig. 5). Reconstituted surfactant adsorbed rapidly, reached a low surface tension on the first and subsequent compressions, and formed very stable films. In vitro cycling did not alter the activity of reconstituted surfactant.


    DISCUSSION
Top
Abstract
Introduction
METHODS
RESULTS
DISCUSSION
References

In a series of publications, Gross and colleagues (7-9) and Krishnasamy et al. (13) reported evidence supporting a model whereby a specific serine hydrolase, termed convertase, promotes the conversion of active apoprotein-rich surfactant subtypes to inactive, less dense, apoprotein-depleted subtypes in a process that depends on a cyclically expanded and compressed surface film. Our laboratory recently proposed the rat serine carboxylesterase ES-2 as a candidate surfactant convertase (1). Although ES-2 is mainly expressed in the liver (22) and secreted into the serum (16), our laboratory was able to demonstrate a lower level of ES-2 expression in the rat lung (1). In the present study, our aim was to establish the cellular sites of ES-2 expression in the lung and to determine whether ES-2 is associated with specific surfactant subtypes.

ES-2 is a member of a family of highly homologous rat serine carboxylesterases. Although several carboxylesterases are present in the lung, the cells responsible for their expression are mostly unknown. ES-2 is distinct from the other cloned carboxylesterases in that it lacks the carboxy-terminal retention sequence common to rat intracellular serine carboxylesterases (20). We exploited this sequence specificity (16) to generate antiserum specific for ES-2 and localized ES-2 to both type II cells and alveolar macrophages in the distal lung. The only other serine carboxylesterase that has been localized to a specific lung cell type is the intracellular esterase ES-10, also called hydrolase A, found in the nonciliated respiratory bronchiolar cell or Clara cell (29). With the use of an antiserum specific for ES-2, our results show a distribution quite different from that described for ES-10. ES-2 is not present in Clara cells but is expressed in alveolar type II cells and alveolar macrophages. Similarly, ES-2 mRNA was localized to type II cells and alveolar macrophages with no detectable signal in Clara cells. With the limited resolution of light microscopy and in situ hybridization alone, it was difficult to distinguish tissue macrophages from type II cells as the source of ES-2 in the alveolar wall. By using a type II cell-specific surface antibody in colocalization experiments, we were able to confirm unambiguously ES-2 expression in type II cells. Our results strongly suggest that ES-2 is synthesized by type II cells and alveolar macrophages in the alveolar space of the lung and document the presence of ES-2 in an appropriate compartment for a role in surfactant homeostasis.

Western blotting of rat BAL fluid and surfactant pellets suggests that most of the enzyme obtained by BAL is not lipid associated. Because ES-2 circulates in the serum, we cannot exclude the possibility that the process of lavage allows additional serum-derived ES-2 to enter the airways and add to the pool of ES-2 derived from lung expression. We did, however, consistently find ES-2 immunoreactivity and nonspecific esterase activity associated with surfactant lipids, with a distribution predominantly restricted to the less dense apoprotein-depleted forms of surfactant. The restricted distribution of ES-2 contrasted with the more diffuse distribution of albumin, a serum protein of similar size, suggesting that the interaction between ES-2 and surfactant lipids was more than a nonspecific interaction or simple entrapment. Our localization of ES-2 mainly to the less dense surfactant fractions is, however, in conflict with data from the mouse where the major diisopropyl fluorophosphate (DFP)-binding protein was localized to the heavy surfactant subtype (6). There are several possible explanations for this apparent discrepancy other than interspecies variation, including differences in the technique of surfactant isolation and esterase detection. The antibody we used was specific for ES-2, whereas the nonspecific inhibitor DFP might have detected other serine hydrolases in mouse surfactant. These results suggest either significant species differences in esterase distribution or the presence of additional DFP-binding proteins in mouse surfactant.

Consistent with previous reports (6, 17), we found that natural rat surfactant containing ES-2 converted to a less dense apoprotein-depleted subtype with in vitro cycling. Cycling also markedly decreased the surface activity of rat surfactant, notably adsorption speed and ability to reach low tensions with repeated compressions. In contrast, surfactant reconstituted from a binary phospholipid mixture and SP-A and SP-B but lacking ES-2 did not convert to a less dense subtype and did not lose SP-B or surface activity with cycling. Although SP-A has been reported to preserve surfactant in a large-aggregate form during cycling (23, 24), this is the first report to characterize the marked resistance to loss of surface activity of surfactants reconstituted with both SP-B and SP-A and lacking endogenous esterase.

Taken together, our results are consistent with a role for ES-2 in subtype conversion in vitro, but we have been unable to purify sufficient ES-2 in an active form to add back to reconstituted surfactant and provide more convincing proof of the hypothesis. Krishnasamy et al. and Dhand et al. recently reported that a crude concanavalin A fraction of mouse BAL fluid containing small amounts of a mouse carboxylesterase (13) increased subtype conversion of reconstituted surfactant containing SP-A and SP-B approximately threefold (4). Conversion was inhibited by heat treatment of the BAL fluid fraction and nonspecific serine hydrolase and esterase and/or lipase inhibitors, suggesting carboxylesterase activity in the concanavalin A fraction may have been responsible for the "convertase" activity (4). This evidence extends our findings and is again supportive of a role for esterase and/or lipase activity in surfactant subtype conversion. Still, a definitive conclusion that a carboxylesterase is responsible for the conversion activity is weakened by the inability to purify ES-2 or its mouse homologue away from other potentially active proteins. The proof of a role for ES-2 in surfactant homeostasis awaits better enzyme purification for the in vitro model systems and complementary studies showing an effect of esterases in surfactant metabolism in vivo.


    ACKNOWLEDGEMENTS

We thank Drs. J. R. Wright and Q. Dong (Duke University, Durham, NC) for advice for the in situ hybridization.


    FOOTNOTES

This work was supported by National Heart, Lung, and Blood Institute Program Project Grant HL-24075 and training grants from the California Lung Association and Wyeth Pediatrics (to H. Clark).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests: S. Hawgood, Cardiovascular Research Institute, Univ. of California, San Francisco, CA 94118-1245.

Received 22 July 1998; accepted in final form 4 November 1998.


    REFERENCES
Top
Abstract
Introduction
METHODS
RESULTS
DISCUSSION
References

1.   Barr, F., H. Clark, and S. Hawgood. Identification of a putative surfactant convertase in rat lung as a secreted serine carboxylesterase. Am. J. Physiol. 274 (Lung Cell. Mol. Physiol. 18): L404-L410, 1998[Abstract/Free Full Text].

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Am J Physiol Lung Cell Mol Physiol 276(3):L452-L458
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