1 Institute for Environmental
Medicine, 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
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.
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
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 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
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 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%
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.
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).
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
-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).
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.
-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.
80°C.
-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.
RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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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.
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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).
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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).
|
To obtain an estimate of the intracellular pool of SP-A receptors,
binding assays were performed on type II cells permeabilized by
exposure to -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
-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.
|
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.
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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).
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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.
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DISCUSSION |
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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 -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.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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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.
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