Mechanism for secretagogue-induced surfactant protein A binding to lung epithelial cells

Qiping Chen1, Aron B. Fisher1, David S. Strayer2, and Sandra R. Bates1

1 Institute for Environmental Medicine, University of Pennsylvania School of Medicine, Philadelphia 19104-6068; and 2 Department of Pathology, Anatomy, and Cell Biology, Jefferson Medical College, Philadelphia, Pennsylvania 19107

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Secretagogues stimulate both secretion and reuptake of surfactant components by pulmonary type II cells as well as enhance surfactant protein A (SP-A) binding. We have evaluated the possibility that the observed increase in SP-A binding is due to the movement of SP-A receptors from an intracellular pool to the plasma membrane. We utilized an anti-idiotypic monoclonal antibody, A2R, which recognizes an SP-A binding protein on type II cell membranes. Immunocytochemistry studies showed that A2R reacted with cellular antigens on type II cell membranes and paranuclear granules. A2R inhibited cell association of 125I-SP-A to type II cells plated on Transwell membranes as well as those plated on plastic dishes and also inhibited the SP-A-stimulated incorporation of phosphatidylcholine liposomes into type II cells. On exposure to secretagogues, the binding of 125I-A2R and 125I-SP-A to type II cells increased in parallel. With permeabilized type II cells on Transwell membranes, one-sixth of the binding sites were located on the plasma membrane, with the remainder being intracellular; phorbol 12-myristate 13-acetate treatment increased the binding of A2R to the cell surface but did not affect the total binding of A2R. Ligand blots of type II cell plasma membranes showed that SP-A and A2R both bound proteins with molecular masses of ~32 and 60 kDa, respectively, reduced. Under nonreducing conditions, the mass of the SP-A and A2R binding protein was ~210 kDa, indicating that the SP-A receptor is composed of disulfide-linked subunits. The results support our hypothesis that secretagogues increase SP-A binding sites by accelerating recruitment of receptors to the cell surface.

surfactant protein A receptors; idiotypic antibody; ligand blot; lung type II cells; surfactant recycling

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

SURFACTANT PROTEIN A (SP-A), the major surfactant-associated protein, has been shown to be a multifunctional protein in the lung. Several studies have demonstrated that SP-A binds with high affinity to type II cells (12, 26). The cell-binding activity of SP-A is responsible for, or directly related to, a number of SP-A functions, including uptake (1, 24, 27) and secretion (7, 18) of phosphatidylcholine (PC) and host defense (17). These functions have been demonstrated with in vitro studies, and their in vivo significance remains to be established.

Several studies have recently identified SP-A binding proteins associated with lung cells. Putative SP-A receptors on type II cell plasma membranes have been characterized by two groups using an idiotypic strategy (19, 20) and by a third group using affinity chromatography (5). Strayer (20) used Curosurf and rabbit surfactant to produce monoclonal antibodies directed against the surfactant components and, using these antibodies as immunogens, generated two monoclonal anti-idiotypic antibodies, A2C and A2R, respectively. On Western blots of pulmonary cells, these anti-idiotypic antibodies reacted strongly with a 30-kDa protein and weakly with additional proteins of 52 and 60 kDa. The cDNA for the 30-kDa protein has been identified and sequenced (22). Stevens et al. (19) produced an auto-anti-idiotypic antibody (2H5) by immunizing mice with human SP-A. This antibody recognizes an SP-A binding protein from type II cells that migrates as 170-200 kDa under nonreducing conditions and ~55 kDa under reducing conditions. The monoclonal anti-idiotypic antibodies 2H5, A2R, and A2C recognize alveolar type II cells by immunocytochemistry, do not react with alveolar macrophages, and inhibit the binding of SP-A to type II cells (19, 21). The functional role of the putative SP-A receptors has been partially addressed. The 30-kDa binding protein is associated with the regulation of secretagogue-stimulated surfactant secretion because the ability of SP-A to block the enhancement of phospholipid secretion from type II cells by secretagogues is interrupted by A2R and A2C (21). The 55-kDa protein may be involved in surfactant endocytosis by type II cells because SP-A-mediated lipid uptake by type II cells is inhibited by the presence of antibody 2H5, whereas this antibody has no effect on surfactant secretion (25). Chroneos et al. (5) purified a novel SP-A receptor from U937 macrophages with a molecular mass of 210 kDa, reduced, using an SP-A affinity column. This receptor has been found in bone marrow-derived macrophages, alveolar macrophages, and alveolar type II epithelial cells. The polyclonal antibody to this receptor was found to block the SP-A-mediated inhibition of phospholipid secretion by type II cells.

The mechanism of SP-A binding to lung type II cells is not completely understood, yet it appears to be critical for the cell-associated functions of SP-A. Previously, Chen et al. (3) reported that secretagogues increase the expression of SP-A receptors on type II cells and that the augmentation in SP-A-receptor number is not dependent on new protein synthesis. They postulated that there is an intracellular pool of SP-A receptors and that exposure to secretagogues leads to accelerated recruitment, with an increase in cell membrane-associated receptor number. By utilizing the anti-idiotypic antibody A2R, the present study further characterizes the type II cell-surface SP-A receptor and examines the response of the receptor to secretagogue stimulation.

    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Cell preparation. Type II cells were isolated from adult male Sprague-Dawley rat lungs according to the procedure of Dobbs et al. (6) as previously described (4). Briefly, after perfusion via the pulmonary artery and lavage through a tracheal cannula, the lungs were digested with elastase and minced in the presence of DNase (Sigma, St. Louis, MO) and fetal bovine serum (ICN Biochemicals, Costa Mesa, CA). The cells were separated by filtration and enriched for type II cells by being plated on rat IgG (Sigma)-coated petri dishes that served to remove most contaminating macrophages. After overnight culture and removal of nonadhered cells, the purity of the type II cell preparation was routinely >90% by modified Papanicolaou stain and the viability was >98% by vital dye exclusion.

Type II cells were plated at 5 × 106 cells on 24-mm inserts of Transwell microporous membranes (3-µm pore size; Costar, Cambridge, MA) or 35-mm plastic tissue culture dishes (Costar) at 3 × 10 6 cells/dish. The cells were cultured in 10% fetal bovine serum in MEM at 37°C in a humidified incubator with 5% CO2 in air. Alveolar macrophages were isolated by centrifugation of rat lung lavage fluid. The purity of the resultant macrophage preparation was >98%.

Purification of SP-A, production of A2R, and iodination. Alveolar lavage fluid was obtained from the Hospital of the University of Pennsylvania and represented therapeutic lavage of lungs of alveolar proteinosis patients. The surfactant was purified with density gradient centrifugation followed by dialysis and lyophilization as previously described (9). SP-A was isolated from the surfactant with 1-butanol and beta -D-glucopyranoside extraction, dialysis, and microconcentration according to the method of Hawgood et al. (11). The purity of the SP-A preparation was monitored with SDS-PAGE according to the method of Laemmli (14) as described previously (1).

The methodology for the production of the anti-idiotypic antibody A2R has been previously described (20). Briefly, monoclonal antibodies to rabbit surfactant were raised in rats. The first screenings of the antibodies indicated that they recognized only a 9-kDa protein (20). However, subsequent screenings revealed that the monoclonal antibody mixture also recognized a 30- to 35-kDa protein. The rats were immunized with a mixture of the anti-rabbit surfactant monoclonal antibodies, and the subsequent anti-idiotypic antisera were screened for the ability to inhibit the binding of rabbit surfactant by the original monoclonal anti-rabbit antibodies. Hybridomas were produced from rats with detectable anti-(anti-rabbit surfactant) titers. The purified anti-idiotypic antibody A2R bound to the alveolar lining and bronchial epithelial cells in lungs and recognized proteins of 30, 52, and 60 kDa in pulmonary cells isolated through vigorous lavage (20) and a 32-kDa protein on plasma membranes from isolated type II cells (21).

SP-A or antibodies were iodinated with Iodo-gen (Pierce, Rockford, IL) according to the directions provided by the manufacturer, and the iodinated protein was dialyzed extensively against Tris buffer. The specific activity and percent trichloroacetic acid precipitability were 512 ± 149 (SD) counts · min-1 (cpm) · ng protein-1 and 96.3 ± 1.7% for SP-A (n = 14 cells), 646 ± 187 cpm/ng protein and 95.6 ± 2.1% for A2R (n = 10 cells), and 462 ± 78 cpm/ng protein and 96.9 ± 0.2% for nonimmune IgG (n = 3 cells), respectively. The iodinated proteins were stored at 4°C and used within 3 wk.

Immunofluorescence. The distribution of the binding sites was investigated in both intact and permeabilized type II cells plated on plastic dishes. Before incubation with the antibody, the cells were either not fixed (intact) or permeabilized by fixation with methanol-acetone (1:1 in volume) for 2 min at 4°C. The intact or permeabilized cells were washed three times with PBS containing 1 mg/ml of BSA (PBS-BSA buffer) and were incubated with nonimmune rat IgG (control) or A2R as a primary antibody at 4°C for 2 h. The cells were washed thoroughly with PBS-BSA buffer and incubated with rhodamine-conjugated goat anti-rat IgG (Sigma) at a dilution of 1:100 in PBS-BSA buffer at 4°C for 1 h. After the cells were washed five times, they were fixed with 4% paraformaldehyde in PBS and examined with a fluorescence microscope.

Incubation of SP-A or antibodies with type II cells. Type II cells plated on plastic dishes or Transwell membranes were cultured for 18 h. Before the start of an experiment, nonadherent cells were removed by washing with MEM. In competition studies to evaluate the effect of antibodies on the cell association of 125I-SP-A with type II cells, the cells were incubated with 0.1 µg/ml of 125I-SP-A and 1 mg/ml of BSA in the presence of 1.0 µg/ml of nonlabeled SP-A or antibody at 37°C for 4 h. For binding assays, type II cells were incubated with or without secretagogues for 20 min at 37°C. Next, the cells were cooled and incubated with iodinated ligands at 4°C for 1 h. In both types of experiments, the nonbound SP-A was removed by three washes with MEM and two washes with PBS. The cells were dissolved in 0.2 N NaOH. The amount of 125I-SP-A bound to the cells was measured with a gamma counter. Background binding by dishes or wells without cells was determined simultaneously, and the amount of radioactivity was subtracted from the samples with cells. The results were normalized to either cellular protein or DNA content.

Preparation of liposomes and uptake by type II cells. Unilamellar liposomes were prepared using lipids with a molar ratio of 0.75 PC [two-thirds dipalmitoyl PC (DPPC) and one-third egg PC] to 0.15 cholesterol to 0.10 egg phosphatidylglycerol (Avanti, Birmingham, AL), with [methyl-3H]choline-labeled DPPC (NEN, Boston, MA) as a tracer. The lipids were dried and resuspended in PBS. Unilamellar liposomes were prepared as previously described (8) by extrusion through a polycarbonate membrane and stored overnight at 4°C. SP-A was added by gentle vortexing and placed on ice for 30 min before use. After incubation of type II cells with the liposomes on Transwell membranes for 1 h at 37°C, the medium was removed, and the cells were washed five times, harvested with 0.05% trypsin, and centrifuged. The cell pellets were recentrifuged once with PBS and sonicated. Aliquots were taken to measure radioactivity and protein (2). Uptake of PC liposomes by the cells was calculated from the [3H]DPPC, which represented two-thirds of the total PC in the liposomes, and the specific activity was adjusted accordingly.

Permeabilization of type II cells. Type II cells were permeabilized according to the procedure of Liu et al. (15). Briefly, the cells plated on Transwell membranes were washed with MEM, and 2.5 ml of permeabilization buffer (MEM containing 40 µM beta -escin) were added for 10 min at 37°C. With this procedure, >90% of type II cells were stained by trypan blue and demonstrated 38% lactate dehydrogenase release (15). The data for this series of experiments are expressed as nanograms of antibody per milligram of DNA because permeabilization of the cells resulted in some loss of protein from the cells but no change in DNA per dish.

Preparation of cell lysate and cell plasma membrane. Freshly isolated type II cells and alveolar macrophages were lysed on ice for 30 min with lysing buffer containing 10 mM imidazole, 2 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml of leupeptin, 1 µg/ml of aprotinin, 1 µg/ml of pepstatin A, and 1 mM benzamidine, pH 7.4, and sonicated on ice for two 15-s cycles. The lysate was centrifuged at 16,000 g for 20 min to separate the total membrane from the cytosol. The total membrane fraction was suspended in 0.32 M sucrose in HEPES-Tris buffer, pH 7.4, layered over a discontinuous sucrose gradient containing 0.5, 0.7, 0.9, and 1.2 M sucrose, and centrifuged at 100,000 g for 1 h at 4°C. The plasma membrane fraction was collected from the 0.9-1.2 M sucrose interface, diluted to 0.32 M sucrose with HEPES-Tris buffer, and centrifuged at 95,000 g for 30 min (9). The plasma membrane pellet was resuspended and stored in aliquots at -80°C.

SDS-PAGE and ligand blotting. The proteins from the particulate membrane or plasma membrane fractions of type II cells were resolved on 10 or 7.5% SDS-PAGE under reducing (5% beta -mercaptoethanol) or nonreducing conditions (14). The proteins were electrophoretically transferred to a nitrocellulose membrane at room temperature overnight (23). The membrane was transiently stained with Ponceau S to monitor the transfer efficiency of the proteins and was blocked with 3-5% Carnation nonfat milk in Tris-buffered saline at room temperature for 45 min on a shaking platform. The nitrocellulose membrane was washed two times with MEM containing 1 mg/ml of BSA and then incubated with 1 µg/ml of labeled ligand in MEM with BSA (1 mg/ml) in a hybridization bag at 4°C for 18 h on a rotating platform. At the end of the incubation, the membranes were washed five times in plastic dishes with MEM containing 1% BSA and two times with PBS. The dried membrane then was exposed to Kodak X-ray film at -80°C. For competition experiments, unlabeled SP-A, A2R, or nonimmunized IgG was added and coincubated with labeled ligand. Quantitation of 125I-SP-A bound to the membrane was accomplished with an Ambis 4000 radioanalytic imager.

Others. Cell protein content was measured by the method of Lowry et al. (16) with BSA as a standard. Cell DNA was measured by a modified Hoechst fluorescence method (13). A preweighed vial of DNA (Sigma) was diluted in the same buffer system and used as a standard in the range of 0-12 µg. Fluorescence was measured in a fluorescence spectrophotometer at 356-nm excitation and 458-nm emission.

Results are reported as means ± SE unless otherwise stated. Results were analyzed statistically by one-way ANOVA with individual comparisons by Dunnett's t-test when appropriate. The level of statistical significance was taken as P < 0.05.

    RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Detection of antibody binding sites by immunocytochemistry. The anti-idiotypic antibody A2R was found to immunostain isolated rat type II cells grown on plastic as previously reported (21). Type II cells cultured overnight on plastic dishes retained their characteristic cytoplasmic structural appearance with lamellar bodies (Fig. 1, A, C, and E). When antibody A2R was reacted with the intact cells at 4°C under conditions where internalization of antibody would not occur, immunofluorescence data demonstrated a reaction primarily at the type II cell surface (Fig. 1B). When the cells were permeabilized, allowing the antibodies to react with both the cell surface and intracellular structures, bright intracellular fluorescence was seen that showed preferential punctate localization to paranuclear granules (Fig. 1D). As a control, little fluorescence was seen in the absence of the primary antibody (Fig. 1F).


View larger version (116K):
[in this window]
[in a new window]
 
Fig. 1.   Immunofluorescent localization of antibody binding to type II cells. A and B: intact cells with anti-idiotypic monoclonal antibody A2R. C and D: permeabilized cells with A2R. E and F: permeabilized cells with nonimmune IgG. A, C, and E: phase micrographs. B, D, and F: fluorescence micrographs. Primary cultures of type II cells on plastic dishes were either not treated (intact) or permeabilized by treatment with methanol-acetone (1:1 in volume). Intact and permeabilized cells were incubated at 4°C for 2 h with either A2R or nonimmune IgG at a concentration of 20 µg/ml, and antibody binding was detected with rhodamine-conjugated goat anti-rat IgG. Bar, 10 µm.

Antibodies inhibit cell association of SP-A to type II cells. It had been previously reported (21) that A2R is an efficient competitor for the binding of iodinated SP-A to type II cells on plastic dishes. To determine whether this antibody altered the binding and uptake of 125I SP-A to type II cells grown on Transwell membranes, competition studies were performed. Type II cells plated on plastic dishes were used for comparative purposes. A2R, nonimmune rat IgG, or unlabeled SP-A (all at 1 µg/ml) were added to type II cells plated on dishes and Transwell membranes. After incubation for 5 min, 125I-SP-A (0.1 µg/ml) was added, and the incubation continued for 4 h at 37°C. Coincubation with unlabeled SP-A under these conditions decreased the type II cell association of 125I-SP-A by ~45% relative to the control values for the cells on both the plastic dishes (Fig. 2A) and Transwell membranes (Fig. 2B). A2R was as effective as unlabeled SP-A in reducing the cell association of 125I-SP-A. Nonimmune IgG had no effect on 125I-SP-A interaction with type II cells on Transwell membranes or plastic dishes.


View larger version (37K):
[in this window]
[in a new window]
 
Fig. 2.   Inhibition of 125I-surfactant protein A (SP-A) cell association to type II cells by unlabeled SP-A or antibodies. Type II cells on plastic dishes (A) and Transwell membranes (B) were preincubated with unlabeled SP-A or antibodies (1.0 µg/ml) at 37°C for 10 min before 0.1 µg/ml of 125I-SP-A was added. Cells were incubated at 37°C for 4 h. Nonbound SP-A was washed off, and cells were dissolved in 0.2 N NaOH. Cell-associated radioactivity was measured. Values are means ± SE of duplicates from 3-5 experiments. Data are presented as a percentage of cell association of 125I-SP-A without additions, which were 7.5 ± 0.8 and 29.0 ± 4.9 ng SP-A/mg cell protein for cells on plastic dishes and Transwell membranes, respectively. * P < 0.05 vs. control cells.

A2R inhibits the SP-A-stimulated uptake of liposomes. SP-A has been shown to have three important effects on the phospholipid turnover of type II cells: downregulation of PC secretion stimulated by secretagogues (7, 18), enhancement of liposome uptake (1, 24, 27), and inhibition of phospholipase A2 (10). The latter effect is directly on the enzyme-substrate interaction and presumably independent of the SP-A receptor. The anti-SP-A-receptor antibody A2R was previously shown to reverse the ability of SP-A to inhibit secretagogue-stimulated phospholipid secretion by type II cells (21). As shown in Table 1, A2R also inhibits the SP-A-mediated promotion of liposome uptake by type II cells. Almost twice as much PC was taken up by the type II cells when the liposomes contained SP-A, whereas the uptake of liposomes with A2R was not different from the control values. Preincubation at 4°C with A2R followed by incubation at 37°C with liposomes containing SP-A abolished the SP-A-stimulated uptake of PC liposomes, whereas preincubation with nonimmune IgG had no effect (Table 1).

                              
View this table:
[in this window]
[in a new window]
 
Table 1.   A2R inhibits SP-A-stimulated liposomal PC uptake

Effect of cAMP or phorbol 12-myristate 13-acetate on the binding of SP-A or anti-idiotypic antibodies. Previously, Chen et al. (3) reported that secretagogues increase the expression of SP-A receptors on type II cells plated on Transwell membranes by demonstrating an increase in cellular 125I-SP-A binding. They postulated that the enhanced SP-A binding was due to an increase in SP-A-receptor number. Thus it would follow that A2R binding to the SP-A receptor also would be enhanced on exposure of the cells to secretagogues. A2R and nonimmune IgG were iodinated and evaluated for their binding to type II cells after secretagogue treatment. On exposure to cAMP (0.1 mM) or phorbol 12-myristate 13-acetate (PMA; 10 nM), type II cells on Transwell membranes increased 125I-SP-A binding by 70%. 125I-A2R binding was also sensitive to secretagogue treatment and increased to a similar extent as SP-A. The cell-surface binding of 125I-nonimmune IgG showed no change in the presence of secretagogues (Fig. 3).


View larger version (24K):
[in this window]
[in a new window]
 
Fig. 3.   Effect of cAMP or phorbol 12-myristate 13-acetate (PMA) on binding of SP-A or antibodies to type II cells plated on Transwell membranes. Type II cells were incubated with cAMP (0.1 mM 8-bromo-cAMP) or PMA (10 nM) at 37°C for 20 min followed by a binding assay where cells were incubated with 125I-SP-A (0.5 µg/ml), 125I-A2R (0.1 µg/ml), or 125I-IgG (0.1 µg/ml) for 1 h at 4°C. Control value was amount of binding for radiolabeled ligand in absence of secretagogues, which were 253 ± 45 ng SP-A, 13.0 ± 1.3 ng A2R, and 14.6 ± 1.2 ng IgG/mg cell protein. Values are means ± SE of duplicates from 3-6 experiments. * P < 0.05 vs. control cells.

To obtain an estimate of the intracellular pool of SP-A receptors, binding assays were performed on type II cells permeabilized by exposure to beta -escin. Initial studies determined that both permeabilized and nonpermeabilized type II cells bound the same amount of iodinated SP-A, possibly because the cell membrane disruptions were too small for SP-A entry. However, the smaller iodinated anti-idiotypic antibody A2R was permeable to beta -escin-treated cells. The binding of 125I-A2R to intact type II cells grown on plastic was 62 ± 5 ng/mg DNA (n = 3; Fig. 4A). Iodinated A2R binding to permeabilized type II cells plated on plastic dishes was 3.3-fold higher than the binding to intact cells. The data confirm the immunocytochemistry observations that visualized intracellular pools of A2R binding sites (Fig. 1D). Intact type II cells plated on Transwell membranes (Fig. 4B) had a 5.6-fold higher binding value for A2R (344 ± 25 ng/mg DNA; n = 3) compared with that of type II cells on plastic dishes. Bates et al. (1) previously showed that iodinated SP-A also binds at a fivefold higher level to type II cells plated on Transwell membranes compared with plastic dishes. Permeabilization of the cells on Transwell membranes resulted in a sixfold greater association of A2R to the cells (Fig. 4B). These results demonstrate that only 16-30% of the total binding sites in the nonpermeabilized type II cells were accessible to the antibody, indicating their localization on the plasma membrane. Because secretagogue treatment had previously been shown to enhance the binding of SP-A to type II cells plated on Transwell membranes while having no affect on cells plated on plastic dishes (3), only type II cells grown on Transwell membranes were exposed to PMA. The data in Fig. 4B show that PMA treatment significantly enhanced A2R binding to intact cells on Transwell membranes but did not change the binding to permeabilized cells, indicating that this secretagogue increased the number of A2R binding sites at the cell surface, whereas the total number of binding sites did not change.


View larger version (18K):
[in this window]
[in a new window]
 
Fig. 4.   125I-A2R binding to type II cells. Type II cells plated on plastic dishes (A) and Transwell membranes (B) were incubated with MEM (intact cells) or 40 µM beta -escin [permeabilized cells (Perm)] at 37°C for 10 min, and binding assay was performed at 4°C with 125I-A2R (0.5 µg/ml) for 2 h. To measure binding to PMA-treated cells, cells were exposed to PMA (10 nM) for 10 min before addition of beta -escin (B). Values are means ± SE of 3 experiments. * P < 0.05 compared with values from intact cells. ** P < 0.05 compared with values from nontreated cells.

Characterization of SP-A receptors by ligand blot. The size of the SP-A and A2R binding proteins was visualized with ligand blots. The proteins from the membrane fraction of type II cells were separated on SDS-polyacrylamide gels run under both reduced and nonreduced conditions, transferred to nitrocellulose paper, and probed with 125I-SP-A or 125I-A2R. Only proteins of ~32 (range 30-35) kDa and 60 (range 55-65) kDa bound to iodinated SP-A in the membrane fraction of type II cells (Fig. 5, lane 1). Quantitation of 125I-SP-A binding to the two protein bands with a radioanalytic imager indicated that 2.2 ± 0.2 (SE) units (n = 5 cell preparations) of SP-A bound to the 32-kDa band for every unit of SP-A bound to the 60-kDa band. The binding of 125I-A2R and 125I-SP-A to type II cell plasma membrane proteins was compared. On the reduced gels (Fig. 5, lanes 1 and 2), iodine-labeled A2R (lane 2) recognized the same molecular-mass proteins of 30-35 and 55-60 kDa as iodinated SP-A (lane 1). On the nonreduced gels with type II cell plasma membranes, iodinated SP-A and A2R (Fig. 5, lanes 3 and 4, respectively) both recognized a protein of ~210 kDa, suggesting that the SP-A binding protein is a multimer composed of disulfide bond-linked proteins of 32 and 60 kDa.


View larger version (57K):
[in this window]
[in a new window]
 
Fig. 5.   Ligand blots of proteins from type II cell plasma membranes. Type II cell plasma membranes were loaded on 10% (left) or 7.5% (right) polyacrylamide gels, 50 µg in each lane, and proteins were separated electrophoretically under reducing (left) or nonreducing (right) conditions. Proteins were transferred, and ligand blots with 125I SP-A (lanes 1 and 3) and 125I-A2R (lanes 2 and 4) were performed as described in METHODS. Nos. on left and right, molecular mass in kDa.

Competition studies showed that the binding of SP-A and A2R to type II cell proteins on the ligand blot was specific. Identical preparations of type II cell plasma membranes were run on SDS-PAGE and transferred to nitrocellulose paper. The ligand blot was performed with iodinated SP-A (0.5 µg/ml) in the absence (control) or presence of unlabeled SP-A or A2R (1 µg/ml). The presence of a twofold excess of unlabeled SP-A or A2R reduced the binding of iodinated SP-A to both the 32- and 60-kDa proteins (Table 2).

                              
View this table:
[in this window]
[in a new window]
 
Table 2.   Competition of 125I-SP-A binding to its receptors by unlabeled SP-A or A2R

Ligand blots of the membrane fraction from alveolar macrophages revealed a different pattern of 125I-SP-A binding compared with type II cells. Two protein bands of ~48 and 58 kDa were the principal proteins visualized, with minor binding to proteins of ~70 and 28 kDa (data not shown). Iodinated nonimmune IgG did not bind to proteins on type II cells membranes. In addition, no visible bands were detected on ligand blots of unlabeled SP-A probed with iodinated SP-A or A2R.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

In a previous study, Chen et al. (3) reported that the secretagogues cAMP and PMA augmented the binding of SP-A to protein sites on type II cells plated on Transwell membranes. They postulated that an accelerated recruitment of SP-A receptors from an intracellular pool would lead to an increase in receptor number on the plasma membranes of the cells, providing more areas for SP-A binding. In the present investigation, we used the anti-idiotypic antibody A2R that recognizes an SP-A binding protein on the surface of type II cells (21). We found that secretagogue treatment enhanced the binding of A2R to type II cells on Transwell membranes to a similar extent as SP-A, indicating that the mechanism for secretagogue enhancement of SP-A binding is through an increase in SP-A-receptor number on the cell surface.

This study provides further evidence that the anti-idiotypic antibody A2R recognizes an SP-A binding protein because coincubation of unlabeled A2R and iodinated SP-A effectively decreased the cell association of SP-A to type II cells grown under two different culture conditions: on plastic dishes and on Transwell membranes. Three possible mechanisms could be involved in the observed inhibition: A2R may bind to the same sites as SP-A, thereby directly competing with SP-A; binding sites for A2R and SP-A may be spatially close to each other and inhibition of SP-A binding would be the result of a stearic effect of A2R binding; and, finally, the binding of A2R to the membrane may result in a conformational change within the SP-A receptor. With secretagogue treatment, we found that the binding of 125I-A2R to type II cells increased to a similar extent as that of 125I-SP-A, indicating that these two ligands share the same receptor. Furthermore, ligand blot analysis with SP-A and A2R identified the same proteins with molecular masses of 30 and 60 kDa, reduced, and 210 kDa, nonreduced. Finally, A2R was able to block two of the biological functions of SP-A, the SP-A-stimulated uptake of liposomes (this study) and the SP-A-induced inhibition of secretagogue-stimulated PC secretion by type II cells (21). From these data, we conclude that SP-A and A2R are interacting with the same protein, a type II cell SP-A receptor.

Two lines of evidence support the existence of intracellular pools of receptors. With the use of immunofluorescent techniques, we found bright localization of A2R at discrete intracellular sites after permeabilization of the type II cells. With beta -escin permeabilization, there was a three- to sixfold greater association of A2R to treated cells than to nonpermeabilized intact cells. These pools would serve as a source for SP-A receptors located on the plasma membrane. The total pool of A2R binding sites, which include the sum of the surface and intracellular components, did not change with PMA treatment, indicating that PMA exposure does not increase the total number of receptor sites. Previously, Chen et al. (3) showed that increased SP-A binding with secretagogue exposure was not altered by inhibition of protein synthesis with cyclohexamide. The data from both studies support the conclusion that new SP-A binding sites are not being produced on secretagogue treatment. In the absence of "new" receptors, the data are consistent with the hypothesis that the increased number of binding sites on the cell surface occurred through recruitment from intracellular pools.

Ligand blots with iodinated SP-A or a modified Western blot with A2R and iodinated Staphyloccocus protein A consistently identified a major protein band of 30-32 kDa in type II cells (20, 21). In addition, two minor protein bands of 52 and 60 kDa were identified in preparations of cell lysates (20). In the present report, we consistently saw iodinated SP-A bind two proteins of ~32 and 60 kDa from the plasma membrane of type II cells. The 60-kDa protein band may represent comigration of the 52- and 60-kDa proteins because a ligand blot of proteins separated by 10% SDS gels may not have resolved two proteins at those molecular masses. On the other hand, the 52-kDa protein may be present only in the cytosol and thus not found in the plasma membrane fractions. Typically, the ratio of 125I-SP-A bound to the 32-kDa protein band to that bound to the 60-kDa protein band in the plasma membranes was 2:1. However, we have noticed variability in the amount of SP-A bound to the 60-kDa protein compared with the 32-kDa protein from sample to sample in the ligand blot, which was not related to amount of protein loading. Although 8 of 12 blots demonstrated that one-third of the total SP-A binding was to the 60-kDa band, three other blots showed only 20% binding and one blot showed 50% binding to the higher molecular-mass band. The reason for the variability is not clear but may be related to the efficiency of transfer of the 60-kDa protein from the gel to the nitrocellulose paper compared with the 32-kDa protein.

SP-A and A2R bound proteins of ~32 and 60 kDa under reducing conditions and 210 kDa under nonreducing conditions. Such data suggest that the SP-A receptor is a multimer composed of 32- and 60-kDa proteins. If we assume that there was an equal transfer efficiency and an equal affinity of A2R and SP-A to both proteins, then the multimer may contain more of the 32-kDa than the 60-kDa component due to the twofold greater binding to the 32-kDa protein band. However, the variability in the ligand blot data precludes definitive quantitation of each component. Using an auto-anti-idiotypic approach, Stevens et al. (19) identified an SP-A receptor of 55 kDa under reducing conditions and 170-200 kDa under nonreducing conditions. In binding assays with type II cells, the auto-anti-idiotypic antibody 2H5 was able to compete with SP-A as has been shown for the anti-idiotypic antibody A2R. Both 2H5 (25) and A2R (this study) antibodies blocked the SP-A-stimulated uptake of phospholipid liposomes. The major difference between the antibodies is that A2R recognizes a 32-kDa protein in addition to the 55- to 60-kDa protein and that A2R blocks the inhibitory effect of SP-A on secretagogue-stimulated PC secretion by type II cells, whereas 2H5 does not. These results raise the possibility that the two antibodies are recognizing different epitopes of the same receptor. The 55-kDa portion of the SP-A receptor may play a role in SP-A-mediated phospholipid uptake, whereas the 32-kDa protein may interact with SP-A in the regulation of surfactant secretion.

The SP-A receptor described here probably differs from the SP-A binding protein described by Chroneos et al. (5), which was originally isolated from macrophages. Polyclonal antibodies raised against this macrophage SP-A binding protein identified a protein of 210 kDa, reduced, with a higher molecular mass under nonreducing conditions. The antibody recognized a 210-kDa protein in type II cell extracts, competed with SP-A for binding to type II cells and macrophages, and inhibited the SP-A effect on PC secretion from type II cells (5). The definitive identity and molecular mass of the various SP-A binding proteins will depend on their isolation and purification.

Because some macrophages contaminate the type II cell preparations, it is important to rule out any contribution of macrophages to the results. Ligand blots of the membrane fraction from macrophages indicated that the principal sites of SP-A binding were to proteins of ~48 and 58 kDa. This binding pattern was not seen in the type II cell plasma membrane preparations. The relationship of these proteins to the 210-kDa macrophage protein cell-surface receptor for SP-A described recently (5) remains to be determined. Significantly, there was no 32-kDa protein bound by SP-A in the macrophage ligand blot, indicating that this protein was specific for type II cell membranes.

Based on these findings and those of the previous study by Chen et al. (3), we conclude that the mechanism for the increase in SP-A binding to type II cells on secretagogue treatment involves the recruitment of SP-A receptors to the cell surface. The data indicate that SP-A receptors are present in the intracellular milieu as well as in the plasma membrane, and a portion of the internal pool is translocated to the surface on exposure to secretagogues. Control of the number and location of SP-A receptors represents a possible mechanism for the regulation of the physiological effects of SP-A.

    ACKNOWLEDGEMENTS

We are grateful to Drs. Paul Lanken and Michael F. Beers for providing lung lavage fluid from alveolar proteinosis patients and to Dr. Henry Shuman and Kathy Notarfrancesco for assistance in the immunocytochemistry studies. We thank Jain-Qin Tao for excellent technical support.

    FOOTNOTES

This research was supported by National Heart, Lung, and Blood Institute Grant HL-19737; National Institute of General Medical Sciences Grant GM-55436; and Council for Tobacco Research Grant 2749A.

Preliminary results of portions of this work were presented at the 1997 American Thoracic Society International Conference in San Francisco, CA, and the 1998 American Thoracic Society International Conference in Chicago, IL.

Address for reprint requests: S. R. Bates, Institute for Environmental Medicine, Univ. of Pennsylvania, 36th and Hamilton Walk, 1 John Morgan Bldg., Philadelphia, PA 19104.

Received 31 October 1997; accepted in final form 2 April 1998.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

1.   Bates, S. R., C. Dodia, and A. B. Fisher. Surfactant protein A regulates uptake of pulmonary surfactant by lung type II cells on microporous membranes. Am J. Physiol. 267 (Lung Cell. Mol. Physiol. 11): L753-L760, 1994[Abstract/Free Full Text].

2.   Bradford, M. M. A rapid and sensitive method for quantitation of microgram quantities of proteins utilizing the principle of protein dye binding. Anal. Biochem. 72: 248-254, 1976[Medline].

3.   Chen, Q., S. R. Bates, and A. B. Fisher. Secretagogues increase the expression of surfactant protein A receptors on lung type II cells. J. Biol. Chem. 271: 25277-25283, 1996[Abstract/Free Full Text].

4.   Chinoy, M. R., C. Dodia, and A. B. Fisher. Increased surfactant internalization by rat type II cells cultured on microporous membranes. Am J. Physiol. 264 (Lung Cell. Mol. Physiol. 8): L300-L307, 1993[Abstract/Free Full Text].

5.   Chroneos, Z., R. Abdolrasulnia, J. A. Whitsett, W. R. Rice, and V. L. Shepherd. Purification of a cell-surface receptor for surfactant protein A. J. Biol. Chem. 271: 16375-16383, 1996[Abstract/Free Full Text].

6.   Dobbs, L. G., R. Gonzalez, and M. Williams. An improved method for isolating type II cells in high yield and purity. Am. Rev. Respir. Dis. 134: 141-145, 1986[Medline].

7.   Dobbs, L. G., J. R. Wright, S. Hawgood, R. Gonzales, K. Venstrom, and J. Nellenbogen. Pulmonary surfactant and its components inhibit secretion of phosphatidylcholine from cultured rat alveolar type II cells. Proc. Natl. Acad. Sci. USA 84: 1010-1014, 1987[Abstract].

8.   Fisher, A. B., and C. Dodia. Role of phospholipase A2 enzymes in degradation of dipalmitoylphosphatidylcholine by granular pneumocytes. J. Lipid Res. 37: 1057-1064, 1996[Abstract].

9.   Fisher, A. B., C. Dodia, and A. Chander. Alveolar uptake of lipid and protein components of surfactant. Am. J. Physiol. 261 (Lung Cell. Mol. Physiol. 5): L334-L340, 1991[Abstract/Free Full Text].

10.   Fisher, A. B., C. Dodia, and A. Chander. Inhibition of lung calcium-independent phospholipase A2 by surfactant protein A. Am. J. Physiol. 267 (Lung Cell. Mol. Physiol. 11): L335-L341, 1994[Abstract/Free Full Text].

11.   Hawgood, S., B. J. Benson, and R. L. Hamilton, Jr. Effects of surfactant-associated proteins and calcium ions on the structure and surface activity of lung surfactant lipids. Biochemistry 24: 184-190, 1985[Medline].

12.   Kuroki, Y., R. J. Mason, and D. R. Voelker. Alveolar type II cells express a high-affinity receptor for pulmonary surfactant protein A. Proc. Natl. Acad. Sci. USA 85: 5566-5570, 1988[Abstract].

13.   Labarca, C., and K. Paigea. A simple, rapid and sensitive DNA assay procedure. Anal. Biochem. 102: 344-352, 1980[Medline].

14.   Laemmli, U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685, 1970[Medline].

15.   Liu, L., M. Wang, A. B. Fisher, and U. P. Zimmerman. Involvement of annexin II in exocytosis of lamellar bodies from alveolar epithelial type II cells. Am. J. Physiol. 270 (Lung Cell. Mol. Physiol. 14): L668-L676, 1996[Abstract/Free Full Text].

16.   Lowry, O. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: 265-275, 1951[Free Full Text].

17.   Pison, U., M. Max, A. Neuendand, S. Weissbach, and S. Peitschmann. Host defence capacities of pulmonary surfactant: evidence for `non-surfactant' functions of the surfactant system. Eur. J. Clin. Invest. 24: 586-599, 1994[Medline].

18.   Rice, W. R., G. F. Ross, F. M. Singleton, S. Kingle, and J. A. Whitsett. Surfactant-associated protein inhibits phospholipid secretion from type II cells. J. Appl. Physiol. 63: 692-698, 1987[Abstract/Free Full Text].

19.   Stevens, P. S., H. Wissel, D. Sieger, V. Meienreis-Sudau, and B. Rustow. Identification of a new surfactant protein A binding protein at the cell membrane of rat type II pneumocytes. Biochem. J. 308: 77-81, 1995[Medline].

20.   Strayer, D. S. Identification of a cell membrane protein that binds alveolar surfactant. Am. J. Pathol. 138: 1085-1095, 1991[Abstract].

21.   Strayer, D. S., B. Pinder, and A. Chander. Receptor-mediated regulation of pulmonary surfactant secretion. Exp. Cell Res. 226: 90-97, 1996[Medline].

22.   Strayer, D. S., S. Yang, and H. H. Jerng. Surfactant protein A-binding proteins. Characterization and structures. J. Biol. Chem. 268: 18679-18684, 1993[Abstract/Free Full Text].

23.   Towbin, H., T. Staehelin, and J. Gordon. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA 76: 4350-4354, 1979[Abstract].

24.   Tsuzuki, A., Y. Kuroki, and T. Akino. Pulmonary surfactant protein A-mediated uptake of phosphatidylcholine by alveolar type II cells. Am. J. Physiol. 265 (Lung Cell. Mol. Physiol. 9): L193-L199, 1993[Abstract/Free Full Text].

25.   Wissel, H., A. C. Looman, I. Friltzsche, B. Rustow, and P. A. Stevens. SP-A-binding protein BP55 is involved in surfactant endocytosis by type II pneumocytes. Am. J. Physiol. 271 (Lung Cell. Mol. Physiol. 15): L432-L440, 1996[Abstract/Free Full Text].

26.   Wright, J. R., J. D. Borchelt, and S. Hawgood. Lung surfactant apoprotein SP-A (26-36 kDa) binds with high affinity to isolated alveolar type II cells. Proc. Natl. Acad. Sci. USA 86: 5410-5414, 1989[Abstract].

27.   Wright, J. R., R. E. Wager, S. Hawgood, L. Dobbs, and J. A. Clements. Surfactant apoprotein Mr = 26,000-36,000 enhances uptake of lipsomes by type II cells. J. Biol. Chem. 262: 2888-2894, 1987[Abstract/Free Full Text].


Am J Physiol Lung Cell Mol Physiol 275(1):L38-L46
0002-9513/98 $5.00 Copyright © 1998 the American Physiological Society