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Receptor-Mediated Regulation of Pulmonary Surfactant Secretion
EXPERIMENTAL CELL RESEARCH ARTICLE NO.
226, 90–97 (1996)
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Receptor-Mediated Regulation of Pulmonary Surfactant Secretion DAVID S. STRAYER,*,1 R. PINDER,*
AND
AVINASH CHANDER†
*Department of Pathology, Anatomy, and Cell Biology and †Department of Pediatrics — Division of Neonatology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107
Surfactant protein A (SP-A) regulates surfactant secretion via an SP-A specific type II cell membrane receptor (SPAR). We report here that two anti-SPAR monoclonal antibodies can modulate the secretory inhibition caused by SP-A. A2C and A2R are rat monoclonal antibodies raised independently and recognize a 32-kDa protein on rat alveolar type II cell membranes. Immunocytochemical studies show that these antibodies bind to isolated type II cells. Scatchard analysis confirms that SP-A binds alveolar type II cells through a single affinity receptor and shows that A2C and A2R recognize that same receptor. Both antibodies inhibit the binding of 125I-SP-A to isolated type II cells. The functional activity of this 32-kDa protein was studied by examining surfactant secretion in isolated type II cells. Surfactant phospholipid secretion was measured in cells that were exposed to various surfactant phospholipid secretagogues (ATP, dibutyryl cAMP, terbutaline, or ionomycin), {SP-A (100 ng/ml), {A2C or A2R. Both antibodies block the negative feedback loop by which SP-A inhibits surfactant secretion. This activity of A2C and A2R is dose-dependent and is independent of the secretagogue used. Thus, the 32kDa type II cell membrane protein bound by A2C and A2R is the functional receptor on alveolar type II cell membranes and regulates type II cell surfactant secretion. q 1996 Academic Press, Inc.
At least four unique proteins (SP-A, -B, -C, and -D) have been isolated from pulmonary surfactant [3]. The most abundant of these is surfactant protein A (SP-A), a heavily glycosylated, apoprotein of É34 kDa under reduced conditions [4, 5]. SP-A serves multiple functions, including stabilizing the structure of intraalveolar tubular myelin [3]. It may also participate in antimicrobial defenses [5]. In addition, SP-A regulates surfactant secretion [6]. Intracellular and extracellular reserves of surfactant are not extensive; so secretion is tightly regulated. It has been shown that SP-A regulates surfactant secretion via a specific type II cell membrane receptor [7, SPAR]. Rice et al. [8] showed that SP-A inhibits surfactant phospholipid secretion. This inhibition is mediated by a specific, high-affinity cell membrane receptor for SP-A [7, 9]. We have identified that receptor [10] and report here on its functional characteristics. Using rat monoclonal anti-idiotype antibodies [11, aiMAbs] raised against monoclonal antibodies that bind surfactant proteins, we identified, structurally characterized, and cloned a É32-kDa protein that binds SP-A and is present on alveolar type II cells. However, the relationship of this protein to surfactant secretion was not clear. We describe here the role of that 32-kDa protein in regulating of surfactant secretion and the ability of the aiMAbs that recognize that protein to prevent the inhibition of surfactant secretion by SP-A.
INTRODUCTION
MATERIALS AND METHODS
Pulmonary surfactant is produced by type II alveolar cells and lowers the surface tension of the air–liquid interface in the alveolar lumen, thus facilitating lung expansion and preventing atelectasis [1]. Chemically, pulmonary surfactant is a complex mixture of É80% glycerophospholipids, É10% cholesterol, and É10% protein. The dipalmitoylated-phosphatidylcholine (DPPC) comprises the largest proportion (É75%) of the lipids found in pulmonary surfactant [2].
Animals. Specific pathogen-free, male Sprague – Dawley rats (180– 200 g) were obtained from Charles River Laboratories. The animals were used as sources of alveolar type II cells within 1 week of receipt. Chemicals and reagents. SP-A from bovine lung was purified and analyzed as described [12, 13]. When labeled SP-A was used, it was labeled with 125I (Dupont-NEN, Wilmington, DE) using chloramineT as described [11]. 125I-SP-A (average specific activity 2 mCi/ug) was purified from unbound iodine using gel filtration chromatography. Isolation of type II cells. Alveolar type II cells were isolated from rat lungs as described by Dobbs and co-workers [14] and modified by Sen and Chander [15]. Essentially, lungs of anesthetized rats were cleared of blood and endotracheally treated with elastase (Worthington Biochemical, Freehold, NJ) to obtain free cells. Once the cells were separated from lung debris, they were plated on bacteriological plates coated with normal rat IgG (Sigma Chemical Co., St
1
To whom correspondence and reprint requests should be addressed at Department of Pathology, Anatomy and Cell Biology, Jefferson Medical College, 1020 Locust St., Philadelphia, PA 19107. Fax: (215)-923-2218. Internet: [email protected]. 90
0014-4827/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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FUNCTIONAL CHARACTERIZATION OF SPAR Louis, MO). After incubation for 1 h, the free cells were collected by ‘‘panning,’’ pelleted by centrifugation, and resuspended (0.6 1 106 cells/ml) in Dulbecco’s minimum essential medium (MEM) containing 10% fetal bovine serum (Gibco-BRL, Gaithersburg, MD). Antibodies. The production and specificities of A2R and A2C rat monoclonal antibodies against the type II cell SPAR have been described [10]. Antibodies were produced by collection of supernatant media from A2C and A2R hybridomas. The aiMAbs A2C and A2R were purified using an affinity chromatography column composed of agarose-bound anti-rat IgG (Sigma Chemical Co.). Bound antibodies were eluted with 100 mM glycine buffer (pH 2.5), neutralized with 1 M Tris (pH 8) buffer, and concentrated using a 30,000 MWCO Centriprep concentrator (Amicon, Inc., Beverly, MA). After concentration, the antibody preparation was desalted by diluting with PBS and reconcentrating using the Centriprep concentrator. The purity of antibodies prepared in this fashion was ascertained by SDS –12% PAGE. Scatchard analysis. Affinity binding studies of SP-A to type II alveolar cells were performed similarly to those of Kuroki et al. [7]. After overnight culture for adherence, type II cells were washed and treated with MEM containing 2 mg/ml BSA plus varying amounts of unlabeled SP-A, A2C, A2R, and/or 125I-SP-A. After incubating a 377C for 4 h, the medium was removed and the cells were washed. The cells were removed from the plates using 2 ml of 0.2 N NaOH. The 125 I-SP-A bound to the cells was measured using a gamma counter (LKB Instruments, Bromma, Sweden). Binding to plates without cells was subtracted from binding data, and data were analyzed [9]. Secretion studies. Secretion studies were performed as described by Sen and Chander [15]. Briefly, 106 alveolar type II cells were cultured for 18 h in MEM containing 10% FBS / 0.5 mCi of [methyl3 H]choline (Amersham, Arlington Heights, IL) to label cellular phospholipids. Cells were washed to remove nonadherent cells and nonincorporated radioactivity. Fresh, serum-free media with the appropriate amount (see Results) of antibody were added to each plate. Plates were incubated for 15 min, after which 100 ng SP-A was added to selected plates, followed by an additional 15 min incubation and then secretagogues in the appropriate concentrations [control (no addition), ATP (1 mM), dibutyryl cAMP (2 mM), terbutaline (100 mM), or ionomycin (1 mM)]. At this point (t Å 0) in each experiment, some samples were removed for analysis of secreted labeled lipids to establish a baseline. Incubation proceeded for an additional 2 h for all other plates. Following this incubation, media were removed and centrifuged (15 min, 300g) to pellet cells that detached during incubation. These cells were later pooled with those recovered from the plates. Lipids from both media and plates were extracted [15] after the addition of [methyl-14C]DPPC (Amersham) as recovery standard and egg phosphatidylcholine (Sigma Chemical Co.) as a carrier lipid. Recovered lipids were dissolved in Scintiverse II fluid (Fisher Scientific, Pittsburgh, PA) and the radioactivity of these lipid extracts was measured with a liquid scintillation counter (Beckmann Instruments, Fullerton, CA). 3H-labeled phospholipid recovery was normalized to recovery of [methyl-14 C]DPPC. Phospholipid secretion was determined as percent secretion Å (CPM in lipids recovered from media 1 100)/(CPM in lipids recovered from media and plates). Western blot analysis. Cell membranes from alveolar type II cells were prepared by treating 107 alveolar type II cells with ice-cold 0.32 M sucrose buffer [10 mM phosphate, 30 mM NaCl, 1 mM MgCl2 , 0.32 M sucrose, and 5 mM phenylmethylsulfonyl fluoride (PMSF)] followed by homogenization and centrifugation (300g, 10 min.). The supernatant was carefully removed and layered over a discontinuous sucrose gradient (1.4 ml of 1.2 M sucrose, followed by 0.7 ml each of 0.9, 0.7, and 0.5 M sucrose) and centrifuged at 95,000g for 60 min. The plasma membrane fraction banded between 0.9 and 1.2 M sucrose. It was diluted to approximately 0.2 M sucrose and centrifuged at 95,000g for 30 min. The resultant pellet was resuspended in 0.32 M sucrose buffer and stored at 0707C until use. Cell membrane proteins were solubilized
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with 1% (final concentration) NP-40 (Sigma Chemical Co.) and separated by SDS–12% PAGE [19]. After electrophoresis, proteins were transferred on to Immobilon-P (Millipore Co., Bedford, MA) as described [16]. Nonspecific binding was blocked with 3% nonfat milk, followed by 20 mg/ml A2C or A2R, and then rabbit anti-rat IgG conjugated to alkaline phosphatase (Sigma Chemical Co.). Filters were washed and binding was visualized by adding the alkaline phosphatase substrates BCIP and NBT (Bio-Rad Laboratories, Inc., Hercules, CA) and allowing formation of a colored precipitate. Immunocytochemistry. Reactivity of these antibodies with isolated rat alveolar type II cells was tested by immunocytochemical analysis. Isolated alveolar type II cells were cultured on glass coverslips and treated with A2C, A2R, or control preparations. Antibody binding was visualized by applying the ABC technique [16]. Diaminobenzidine and H 2O 2 were added to elicit a colored reaction product and slides were examined using an Olympus photomicroscope.
RESULTS
Antibodies Bind to Type II Cells Previous work [10, 11] showed that A2C and A2R, two independently raised aiMAbs, bound a É32-kDa protein in lung homogenates and that the protein they recognized was present at the cell membrane of human, rabbit, and pig alveolar type II cells. However, since functional studies of surfactant secretion are performed with rat alveolar type II cells it is important to ascertain that these rat aiMAbs bind to rat alveolar type II cells. The alveolar type II cell isolation procedure used here, provides preparations that are ú95% type II cells, as determined by fluorescence staining with phosphine 3R [14, 15]. To test whether these type II cells compare to human and other species’ type II cells, which are recognized by A2R and A2C, immunocytochemical staining showed that both A2C and A2R bind to isolated type II cells, at the cell membrane (Fig. 1). We had previously applied Western and ligand blot analyses to determine that A2C and A2R bind a 32-kDa type II cell membrane protein from human and porcine lungs and that this 32-kDa protein also binds SP-A. Therefore, Western blot and ligand blot analyses were used to analyze isolated cell membrane proteins from these enriched rat type II cell preparations. This analysis showed that A2C and A2R bind a single 32-kDa rat alveolar type II cell membrane protein and that a protein of the same size also binds SP-A (Fig. 2). Antibodies Bind to a Receptor and Competitively Inhibit SP-A Binding To ascertain that these type II cell preparations are comparable to those reported by others to study SP-A receptor activity, we measured SP-A receptor number and affinity in our cell preparations. The SP-A binding characteristics of these isolated type II cells were studied by Scatchard analysis. Binding of SP-A to these type II cells was determined (Fig. 3a). We found that type II cell SP-A binding was maximal at 2 mg/ml SPA. Alveolar type II cells were incubated with various
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concentrations of 125I-SP-A, and the free and bound pools were measured [7]. We observed a monophasic SP-A binding curve (Figs. 3a and 3b), indicating that the SP-A receptors on our type II cell preparations bind SP-A with uniform affinity (Kd Å 1.20 { 0.13 mg/ml) and that there are 105 SP-A binding molecules for each type II cell. The shape of the binding curve, indicating positive cooperativity, and the Kd and receptor numbers are virtually identical to those reported by Kuroki et al. [7], indicating that our type II cell preparations are comparable to those reported by others. To characterize further the reactivity of A2C and A2R with type II cell membrane proteins, the inhibition of SP-A binding by these aiMAbs was examined. Iodinated SP-A (5 mg/ml) and various concentrations (between 0 and 10 mg/ml) of unlabeled SP-A, A2C, and A2R were added as potential competitive inhibitors of the SPAR – 125I-SP-A interaction. These inhibitors were incubated with the alveolar type II cells for 4 h. Both antibodies reduce binding of iodinated SP-A to isolated alveolar type II cells (Fig. 3c). The reduction in binding of 125I-SP-A to alveolar type II cells was dose-dependent. At high concentrations of inhibitor, the percentages of inhibition were comparable for A2R, A2C, and unlabeled competing SP-A. Furthermore, maximal reduction of 125I-SP-A binding was seen with 1 mg/ml of A2C, 0.5 mg/ml of A2R, and 10 mg/ml of SP-A.
dependent fashion (P õ 1004 by x2 analysis). Maximal reversal of inhibition was observed at A2C concentrations ú 50 mg/ml, but statistically highly significant increases in secretion occurred at A2C concentrations §1 mg/ml. Normal rat IgG had no effect on SP-A inhibition of phospholipid secretion. These results are consistent with the conclusion that A2C (and A2R) binds to an SP-A receptor and blocks its inhibitory function. The dose–response curves shown in Fig. 4 were produced using isolated alveolar type II cells treated with ATP. A number of compounds are known to stimulate surfactant secretion [2]. SP-A inhibits the action of these secretagogues [8]. However, these secretagogues act through different mechanisms. Thus, it is important to determine whether A2C and A2R also reverse the SP-A induced inhibition of surfactant secretion elicited by other secretagogues. The secretagogues tested (ATP, dibutyryl cAMP, terbutaline, and ionomycin) were selected to represent the variety of modulators known to stimulate surfactant secretion. Regardless of the secretagogue used, alveolar type II cells incubated with 100 ng exogenous SP-A secreted less phospholipid than did cells incubated without SP-A (Fig. 5). A2C reversed the inhibition of secretion by SP-A regardless of the secretagogue, although in some cases A2C did not restore surfactant secretion completely (Fig. 5).
A2R and A2C Reverse SP-A Inhibition of Type II Cell Phospholipid Secretion
DISCUSSION
Anti-idiotype MAbs A2C and A2R bind a É32-kDa type II cell membrane protein and inhibit the binding of SP-A to isolated alveolar type II cells. The function of this SP-A binding protein remained to be determined. Thus, we tested whether this É32-kDa protein also performed the recognized SP-A receptor function for type II cells: regulation of surfactant secretion. When isolated alveolar type II cells are incubated with SP-A, they do not secrete as much phospholipid in response to various secretagogues (e.g., ATP) as do cells incubated without SP-A [8]. To determine whether the É32-kDa protein bound by A2C and A2R is a functional receptor, we preincubated alveolar type II cells with [methyl3 H]choline to label cellular lipids and then treated them with antibody or normal IgG, followed by SP-A (100 ng/ml) and ATP (1 mM). This dose of SP-A blocked type II cell phospholipid secretion. Antibodies A2C (Fig. 4) and A2R (not shown) reversed this inhibition in a dose-
Surfactant secretion by alveolar type II cells is regulated by complex mechanisms that respond to a wide variety of stimuli. Both physical (e.g., deep inflation) and chemical (e.g., purines, adrenergic agonists, and calcium ionophores) stimuli reportedly affect surfactant secretion (reviewed, ref. 2). The means by which physical stimuli increase surfactant secretion are unknown. The cytokines and biologically active chemicals that are surfactant secretagogues may act through at least three different intracellular signaling systems (e.g., increasing Ca2/ and cAMP and activating PKC). The mechanisms by which these various secretagogues modulate surfactant secretion are being elucidated and are still unclear. However, very little is known about the regulation of surfactant secretion by SP-A. Surfactant secretion is regulated by a negative feedback loop in which SP-A, a protein produced by alveolar type II cells, binds to a specific receptor on type II cell mem-
FIG. 1. A2C and A2R antibodies recognize a rat type II cell membrane protein. Type II alveolar pneumocytes were isolated according to [14] and allowed to adhere as usual. The cultures were fixed with acetone, then treated with affinity-purified rat monoclonal anti-idiotype antibody A2R (a, 11100), A2C (b, 11100), or normal rat IgG (c, 1600) (original magnifications). Antibody binding was visualized by the ABC technique (see Materials and Methods). Slides were counterstained with hematoxylin. The aiMAb bound at the plasma membrane of isolated rat type II cells.
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FIG. 2. SP-A and anti-idiotype monoclonal antibodies A2R and A2C bind a 32-kDa rat type II cell membrane protein. Type II alveolar pneumocytes were prepared as usual. Cell membranes were isolated from these cells according to standard procedures (see Materials and Methods), solubilized, separated by SDS– PAGE, and electroblotted to PVDF membranes. These membranes were then exposed to affinity-purified A2C or A2R, followed by rabbit anti-rat IgG then 125I-Staph. protein A. Alternatively, the membranes were exposed to 125I-labeled SP-A (10). The locations of coelectrophoresed molecular size markers are shown at the right. SP-A, A2C, and A2R all bound a 32-kDa protein from rat type II cell membranes.
branes [7–11]. This interaction reduces the rate of surfactant secretion. The identification and characterization of the SP-A receptor has proven difficult. However, we developed two monoclonal aiMAbs for this purpose. The aiMAbs, A2C and A2R, were derived independently by immunizing different rats with different combinations of monoclonal antibodies against surfactant protein. Hybridomas were selected for their ability to block the ability of antibodies against surfactant proteins to bind surfactant protein-A. A2C and A2R bound a É30-kDa non-Ig protein on rabbit, pig, and human lung cell membranes [11]. We have reported the use of these aiMAbs to identify human and porcine SP-A binding proteins in lung cDNA expression libraries and described the structure of these proteins and their cDNAs [10]. The use of aiMAbs to identify an unknown or putative receptor protein is well established. The theoretical basis for this approach was described by Jerne [17]. Used in situations in which a ligand is available and antibodies to the ligand are on hand, the practical application of anti-idiotype antibody approaches for the identification of cell membrane receptors has been amply demonstrated [18–20]. Most recently, this technique has received molecular substantiation with an X-ray crystallographic demonstration that receptor – anti-idiotype interaction may closely resemble, spatially, the receptor–ligand interaction, even when the anti-idiotype and the ligand appear to be much different from each other [21]. We report here that the aiMAbs A2C and A2R bind a É32-kDa non-Ig protein on rat alveolar type II cells.
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This finding is important for several reasons. Our previous reports that A2R and A2C recognized a É30-kDa SP-A binding protein were based on Western blot analysis of whole lung homogenates, immunohistochemical localization in tissue sections, and both Western and ligand blot studies of recombinant human and porcine SP-A binding proteins. The present report uses isolated type II cells to establish the cell-type specificity and the subcellular localization of the SP-A binding protein, i.e., alveolar type II cell membranes. The present work confirms the molecular size of this protein of É32-kDa, which is consistent with the deduced open reading frame sizes for both human and pig SP-A binding protein [10]. The demonstrable reactivity of A2R and A2C with type II cells from the same animal species that produced these aiMAbs (rat) further confirms that type II cells are the targets to which A2C and A2R bind and that the É32-kDa protein recognized by A2C and A2R is a type II cell membrane protein. The data presented here establish the role of this É32-kDa SP-A binding protein as a functional SP-A receptor (SPAR). A2C and A2R interrupt the negative feedback loop that is used by alveolar type II cells to regulate surfactant secretion. Surfactant secretion is stimulated by a number of secretagogues and SP-A inhibits surfactant secretion regardless of the secretagogue used [8]. Thus, the regulatory activity of any putative SP-A receptor should be demonstrably impervious to the variety of secretagogues used. The É32kDa protein recognized by A2C and A2R fits this requirement: A2C and A2R both restore the ability of alveolar type II cells to secrete surfactant in the presence of inhibiting amounts of SP-A regardless of the secretagogue used (ATP, dibutyryl-cAMP, terbutaline, or ionomycin). In their analysis of SP-A receptor avidity and cell membrane density, Kuroki et al. reported additional studies and applied corrections in their calculations, to compensate for SP-A internalization during the study period [7]. Our studies, which were performed in the presence of excess labeled SP-A, were intended to ascertain that our type II cell preparations were comparable to those studied by others in terms of SP-A binding, receptor number, and receptor affinity. Our quantitative binding studies showed that 105 SP-A molecules bound each type II cell and that the affinity of this binding was 1.2 { 0.13 mg/ml. These numbers are comparable to previously reported data (1.35 1 10 5 SP-A molecules bound/cell, Kd Å 1.02 { 0.32 mg/ml [7]). The Scatchard plot of these data is very similar to that reported by Kuroki et al. [7] and shows positive cooperativity in binding. We found that A2R and A2C inhibited SP-A binding to this receptor with similar affinity. These data confirm that A2C and A2R bound the same molecule that binds SP-A and indicate that the ability of these ai-
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FIG. 3. A2C and A2R inhibit SP-A binding to rat alveolar type II cells. Type II alveolar pneumocytes were isolated as according to [14] and cultured overnight to allow attachment to plastic tissue culture plates. The following day, the plates were washed and then treated with fresh medium. (a) Binding of 125 I-SP-A to type II cells was measured as a function of the 125I-SP-A concentration. For Scatchard analysis (b) various concentrations of 125I-SP-A were added, while for analysis of inhibition (c) a constant concentration (5 mg/ml) of 125I SPA and various amounts of unlabeled SP-A, A2C, or A2R. The cells were incubated for 4 h, and the pools of free and bound 125I SP-A were measured as described by Kuroki et al. [7]. Results shown are the means ({SEM) of at least two independent determinations, each with §2 replicates. 125I SP-A binding is similar to that described by Kuroki et al. Furthermore, both A2C and A2R inhibit the binding of 125I SPA in a dose-dependent fashion. Differences between the conditions of 0.01 mg/ml inhibitor on the one hand and all of the antibody concentrations tested on the other; and between SP-A as an inhibitor on the one hand and the antibodies on the other, at concentrations between 0.01 and 1.0 mg/ml are statistically significant (P õ 0.05).
MAbs to interrupt the SP-A –surfactant secretion negative feedback loop may reflect, in part, their ability to displace SP-A from SPAR. A2R and A2C displace 125ISP-A from SPAR more efficiently than does unlabeled SP-A at low concentrations, but comparably to unlabeled SP-A at high concentrations in the same assay. The data presented here, taken together with our previously published reports, support the conclusion that the É32-kDa protein recognized by A2C and A2R is the functional receptor for alveolar type II cells. SP-
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A inhibits surfactant secretion. A2C and A2R, but not nonspecific IgG, reverse this inhibition. A2C and A2R bind one alveolar type II cell membrane protein. Scatchard analysis shows that SP-A binding activity is present on type II cells and that A2C, A2R, and SP-A all bind this receptor in similar fashion. The mechanism by which this SP-A receptor affects surfactant secretion is not understood. One possibility is that even though different secretagogues may generate different intracellular signals and act through
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FIG. 4. A2C antibody restores surfactant secretion by rat type II cells. Type II alveolar pneumocytes were isolated according to [14] and cultured overnight with [methyl-3H]choline as usual. The following day, cultures were treated with ATP, followed by different concentrations of A2C or normal rat IgG, and 100 ng/ml purified bovine SP-A. Surfactant secretion is measured as the percentage of total phospholipid that is secreted within 2 h. Results shown are the means ({SEM) of at least three different determinations. The range (mean { SEM) of surfactant secretion by isolated type II cells not treated or treated only with ATP is shown. Surfactant secretion by isolated type II cells treated with ATP and SP-A was 0.62 { 0.09. The A2C and IgG dose-response curves shown are significantly different from each other by x2 analysis (P ! 0.01). Normally, SP-A inhibits surfactant secretion. A2C, but not normal IgG, restored surfactant secretion by type II cells in a dose-dependent fashion.
mechanisms that are initially different, these diverse signals activate a single pathway of surfactant secretion. SPAR regulates the activity of this distal pathway regardless of the means used to enhance secretion.
SPAR cDNA bears some homology to other (e.g., ryanodine) receptor proteins, in particular cDNAs encoding receptors that are linked to a Ca 2/ second messenger system [10]. Therefore, it is possible SPAR activa-
FIG. 5. Effects of SP-A and A2C on surfactant secretion by rat type II cells stimulated by different secretagogues. Type II pneumocytes were isolated and cultured overnight with [methyl- 3H]choline to label phospholipids as usual. The following day, cultures were treated with one of several different secretagogues: ATP, terbutaline, cAMP, or ionomycin, {5 mg/ml A2C, {100 ng/ml SP-A. Surfactant secretion was measured after 2 h. Results shown are the means ({SEM) of at least three different determinations. SP-A inhibits the surfactant secretion that is stimulated by each of the different secretagogues tested. In all cases, A2C reversed this inhibition and restored surfactant secretion. * P õ 0.02 compared to parallel cultures receiving secretagogue only and compared to parallel cultures receiving secretagogue / SP-A / A2C. ** P õ 0.02 compared to parallel cultures treated with ATP, but without A2C or SP-A. All other groups are statistically indistinguishable from the secretagogue alone group for each individual secretagogue, respectively.
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tion regulates cellular Ca2/ traffic. The involvement of Ca2/ in regulation of surfactant secretion has been suggested previously. A number of surfactant secretagogues (i.e., ATP, ionomycin, and terbutaline) cause transient increases in intracellular Ca2/ of alveolar type II cells [22]. Furthermore, surfactant secretion can be inhibited by incubating alveolar type II cells in the presence of W-7, a known calmodulin blocker [23]. Binding of SP-A to SPAR may potentially inhibit these Ca2/ transients because alveolar type II cells incubated in the presence of SP-A / ionomycin had lower intracellular Ca2/ concentrations than did cells treated with ionomycin alone [24]. In the present experiments SPA inhibited ionomycin-stimulated surfactant secretion, and A2C reversed the SP-A induced inhibition, suggesting that SPAR may modulate surfactant secretion by regulating cytosol calcium. The present studies indicate that these antibodies may potentially be used to treat diseases of surfactant insufficiency, including neonatal and adult respiratory distress syndromes (RDS). Treatment for these diseases currently involves administration of surfactants extracted from animal lungs or prepared artificially. While quite useful for neonatal RDS, surfactant replacement therapy is only being applied experimentally to adult RDS and is quite costly. These antibodies may offer an additional, perhaps supplemental, approach to stimulate secretion of endogenous surfactant. The work presented heretofore would not have been possible without the kind technical help of Ricardo Moraes, Ai-Min Wu, and Meng Zhao. In addition, the support and advice of the following investigators were helpful in our studies: Sandra Bates, Michael Beers, Aron Fisher, Simon Slater, Andrew Thomas, Dennis Voelker, and Jeff Whitsett. Immunocytochemical studies were kindly performed by Al Kovatich and Mike Noble, under the direction of Dr. Roland Schwarting. This work was supported by Grant 2749A from the Council for Tobacco Research, U.S.A., as well as by NIH Grants AA7463 and HL49959.
REFERENCES 1.
Farrell, P. M., and Avery, M. E. (1975) Am. Rev. Respir. Dis. 111, 657–688.
2. Chander, A., and Fisher, A. B. (1990) Am. J. Physiol. 258, L241 – L253. 3. Hawgood, S., and Shiffer, K. (1991) Annu. Rev. Physiol. 53, 375–394. 4. Hawgood, S., Efrati, H., Schilling, J., and Benson, B. J. (1985) Biochem. Soc. Trans. 13, 1092 –1096. 5. Eijking, E. P., van Daal, G. J., Tenbrinck, R., Sluiters, J. F., Hannappel, E., Erdmann, W., and Lachmann, B. (1992) Adv. Exp. Med. Biol. 316, 293–298. 6. Dobbs, L. G., Wright, J. R., Hawgood, S., Gonzalez, R., Venstrom, K., and Nellenbogen, J. (1987) Proc. Natl. Acad. Sci. USA 84, 1010 –1014. 7. Kuroki, Y., Mason, R. J., and Voelker, D. R. (1988) Proc. Natl. Acad. Sci. USA 85, 5566 –5570. 8. Rice, W. R., Ross, G. F., Singleton, F. M., Dingle, S., and Whitsett, J. A. (1987) J. Appl. Physiol. 63, 692 –698. 9. Kuroki, Y., Mason, R. J., and Voelker, D. R. (1988) J. Biol. Chem. 263, 17596 –17602. 10. Strayer, D. S., Yang, S., and Jerng, H. H. (1993) J. Biol. Chem. 268, 18679 –18684. 11. Strayer, D. S. (1991) Am. J. Pathol. 138, 1085 –1095. 12. Fisher, A. B., Arad, I., Dodia, C., Chander, A., and Feinstein, S. I. (1991) Am. J. Physiol. 260, L226 –L233. 13. Hawgood, S., Benson, B. J., and Hamilton, Jr., R. L. (1985) Biochemistry 24, 184– 190. 14. Dobbs, L. G., Gonzalez, R., and Williams, M. C. (1986) Am. Rev. Respir. Dis. 134, 141–145. 15. Sen, N., and Chander, A. (1994) Biochem. J. 298, 681– 687. 16. Hsu, S. M., Raine, L., and Fanger, H. (1981) J. Histochem. Cytochem. 29, 577–580. 17. Jerne, N. K. (1974) Ann. Immun. Inst. Pasteur 125C, 373–389. 18. Nisonoff, A., and Lamoyi, E. (1981) Clin. Immun. Immunopathol. 21, 397– 406. 19. Greene, M. I., and Co, M. S. (1987) Monogr. Allergy 22, 120– 125. 20. Strayer, D. S., Vitetta, E. S., and Kohler, H. (1975) J. Immunol. 114, 722– 727. 21. Fields, B. A., Goldbaum, F. A., Ysern, X., Poljak, R. J., and Marluzza, R. A. (1995) Nature 374, 739–742. 22. Sano, K., Voelker, D. R., and Mason, R. J. (1987) Am. J. Physiol. 253, C679 –C686. 23. Voyno-Yasetnetskaya, T. A., Dobbs, L. G., and Williams, M. C. (1991) Am. J. Physiol. 261(Suppl.), 105–109. 24. Pian, M. S., and Dobbs, L. G. (1988) FASEB Proc. 2, A957. [Abstract]
Received February 12, 1996 Revised version received April 18, 1996
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Receptor-Mediated Regulation of Pulmonary Surfactant Secretion DAVID S. STRAYER,*,1 R. PINDER,*
AND
AVINASH CHANDER†
*Department of Pathology, Anatomy, and Cell Biology and †Department of Pediatrics — Division of Neonatology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107
Surfactant protein A (SP-A) regulates surfactant secretion via an SP-A specific type II cell membrane receptor (SPAR). We report here that two anti-SPAR monoclonal antibodies can modulate the secretory inhibition caused by SP-A. A2C and A2R are rat monoclonal antibodies raised independently and recognize a 32-kDa protein on rat alveolar type II cell membranes. Immunocytochemical studies show that these antibodies bind to isolated type II cells. Scatchard analysis confirms that SP-A binds alveolar type II cells through a single affinity receptor and shows that A2C and A2R recognize that same receptor. Both antibodies inhibit the binding of 125I-SP-A to isolated type II cells. The functional activity of this 32-kDa protein was studied by examining surfactant secretion in isolated type II cells. Surfactant phospholipid secretion was measured in cells that were exposed to various surfactant phospholipid secretagogues (ATP, dibutyryl cAMP, terbutaline, or ionomycin), {SP-A (100 ng/ml), {A2C or A2R. Both antibodies block the negative feedback loop by which SP-A inhibits surfactant secretion. This activity of A2C and A2R is dose-dependent and is independent of the secretagogue used. Thus, the 32kDa type II cell membrane protein bound by A2C and A2R is the functional receptor on alveolar type II cell membranes and regulates type II cell surfactant secretion. q 1996 Academic Press, Inc.
At least four unique proteins (SP-A, -B, -C, and -D) have been isolated from pulmonary surfactant [3]. The most abundant of these is surfactant protein A (SP-A), a heavily glycosylated, apoprotein of É34 kDa under reduced conditions [4, 5]. SP-A serves multiple functions, including stabilizing the structure of intraalveolar tubular myelin [3]. It may also participate in antimicrobial defenses [5]. In addition, SP-A regulates surfactant secretion [6]. Intracellular and extracellular reserves of surfactant are not extensive; so secretion is tightly regulated. It has been shown that SP-A regulates surfactant secretion via a specific type II cell membrane receptor [7, SPAR]. Rice et al. [8] showed that SP-A inhibits surfactant phospholipid secretion. This inhibition is mediated by a specific, high-affinity cell membrane receptor for SP-A [7, 9]. We have identified that receptor [10] and report here on its functional characteristics. Using rat monoclonal anti-idiotype antibodies [11, aiMAbs] raised against monoclonal antibodies that bind surfactant proteins, we identified, structurally characterized, and cloned a É32-kDa protein that binds SP-A and is present on alveolar type II cells. However, the relationship of this protein to surfactant secretion was not clear. We describe here the role of that 32-kDa protein in regulating of surfactant secretion and the ability of the aiMAbs that recognize that protein to prevent the inhibition of surfactant secretion by SP-A.
INTRODUCTION
MATERIALS AND METHODS
Pulmonary surfactant is produced by type II alveolar cells and lowers the surface tension of the air–liquid interface in the alveolar lumen, thus facilitating lung expansion and preventing atelectasis [1]. Chemically, pulmonary surfactant is a complex mixture of É80% glycerophospholipids, É10% cholesterol, and É10% protein. The dipalmitoylated-phosphatidylcholine (DPPC) comprises the largest proportion (É75%) of the lipids found in pulmonary surfactant [2].
Animals. Specific pathogen-free, male Sprague – Dawley rats (180– 200 g) were obtained from Charles River Laboratories. The animals were used as sources of alveolar type II cells within 1 week of receipt. Chemicals and reagents. SP-A from bovine lung was purified and analyzed as described [12, 13]. When labeled SP-A was used, it was labeled with 125I (Dupont-NEN, Wilmington, DE) using chloramineT as described [11]. 125I-SP-A (average specific activity 2 mCi/ug) was purified from unbound iodine using gel filtration chromatography. Isolation of type II cells. Alveolar type II cells were isolated from rat lungs as described by Dobbs and co-workers [14] and modified by Sen and Chander [15]. Essentially, lungs of anesthetized rats were cleared of blood and endotracheally treated with elastase (Worthington Biochemical, Freehold, NJ) to obtain free cells. Once the cells were separated from lung debris, they were plated on bacteriological plates coated with normal rat IgG (Sigma Chemical Co., St
1
To whom correspondence and reprint requests should be addressed at Department of Pathology, Anatomy and Cell Biology, Jefferson Medical College, 1020 Locust St., Philadelphia, PA 19107. Fax: (215)-923-2218. Internet: [email protected]. 90
0014-4827/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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FUNCTIONAL CHARACTERIZATION OF SPAR Louis, MO). After incubation for 1 h, the free cells were collected by ‘‘panning,’’ pelleted by centrifugation, and resuspended (0.6 1 106 cells/ml) in Dulbecco’s minimum essential medium (MEM) containing 10% fetal bovine serum (Gibco-BRL, Gaithersburg, MD). Antibodies. The production and specificities of A2R and A2C rat monoclonal antibodies against the type II cell SPAR have been described [10]. Antibodies were produced by collection of supernatant media from A2C and A2R hybridomas. The aiMAbs A2C and A2R were purified using an affinity chromatography column composed of agarose-bound anti-rat IgG (Sigma Chemical Co.). Bound antibodies were eluted with 100 mM glycine buffer (pH 2.5), neutralized with 1 M Tris (pH 8) buffer, and concentrated using a 30,000 MWCO Centriprep concentrator (Amicon, Inc., Beverly, MA). After concentration, the antibody preparation was desalted by diluting with PBS and reconcentrating using the Centriprep concentrator. The purity of antibodies prepared in this fashion was ascertained by SDS –12% PAGE. Scatchard analysis. Affinity binding studies of SP-A to type II alveolar cells were performed similarly to those of Kuroki et al. [7]. After overnight culture for adherence, type II cells were washed and treated with MEM containing 2 mg/ml BSA plus varying amounts of unlabeled SP-A, A2C, A2R, and/or 125I-SP-A. After incubating a 377C for 4 h, the medium was removed and the cells were washed. The cells were removed from the plates using 2 ml of 0.2 N NaOH. The 125 I-SP-A bound to the cells was measured using a gamma counter (LKB Instruments, Bromma, Sweden). Binding to plates without cells was subtracted from binding data, and data were analyzed [9]. Secretion studies. Secretion studies were performed as described by Sen and Chander [15]. Briefly, 106 alveolar type II cells were cultured for 18 h in MEM containing 10% FBS / 0.5 mCi of [methyl3 H]choline (Amersham, Arlington Heights, IL) to label cellular phospholipids. Cells were washed to remove nonadherent cells and nonincorporated radioactivity. Fresh, serum-free media with the appropriate amount (see Results) of antibody were added to each plate. Plates were incubated for 15 min, after which 100 ng SP-A was added to selected plates, followed by an additional 15 min incubation and then secretagogues in the appropriate concentrations [control (no addition), ATP (1 mM), dibutyryl cAMP (2 mM), terbutaline (100 mM), or ionomycin (1 mM)]. At this point (t Å 0) in each experiment, some samples were removed for analysis of secreted labeled lipids to establish a baseline. Incubation proceeded for an additional 2 h for all other plates. Following this incubation, media were removed and centrifuged (15 min, 300g) to pellet cells that detached during incubation. These cells were later pooled with those recovered from the plates. Lipids from both media and plates were extracted [15] after the addition of [methyl-14C]DPPC (Amersham) as recovery standard and egg phosphatidylcholine (Sigma Chemical Co.) as a carrier lipid. Recovered lipids were dissolved in Scintiverse II fluid (Fisher Scientific, Pittsburgh, PA) and the radioactivity of these lipid extracts was measured with a liquid scintillation counter (Beckmann Instruments, Fullerton, CA). 3H-labeled phospholipid recovery was normalized to recovery of [methyl-14 C]DPPC. Phospholipid secretion was determined as percent secretion Å (CPM in lipids recovered from media 1 100)/(CPM in lipids recovered from media and plates). Western blot analysis. Cell membranes from alveolar type II cells were prepared by treating 107 alveolar type II cells with ice-cold 0.32 M sucrose buffer [10 mM phosphate, 30 mM NaCl, 1 mM MgCl2 , 0.32 M sucrose, and 5 mM phenylmethylsulfonyl fluoride (PMSF)] followed by homogenization and centrifugation (300g, 10 min.). The supernatant was carefully removed and layered over a discontinuous sucrose gradient (1.4 ml of 1.2 M sucrose, followed by 0.7 ml each of 0.9, 0.7, and 0.5 M sucrose) and centrifuged at 95,000g for 60 min. The plasma membrane fraction banded between 0.9 and 1.2 M sucrose. It was diluted to approximately 0.2 M sucrose and centrifuged at 95,000g for 30 min. The resultant pellet was resuspended in 0.32 M sucrose buffer and stored at 0707C until use. Cell membrane proteins were solubilized
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with 1% (final concentration) NP-40 (Sigma Chemical Co.) and separated by SDS–12% PAGE [19]. After electrophoresis, proteins were transferred on to Immobilon-P (Millipore Co., Bedford, MA) as described [16]. Nonspecific binding was blocked with 3% nonfat milk, followed by 20 mg/ml A2C or A2R, and then rabbit anti-rat IgG conjugated to alkaline phosphatase (Sigma Chemical Co.). Filters were washed and binding was visualized by adding the alkaline phosphatase substrates BCIP and NBT (Bio-Rad Laboratories, Inc., Hercules, CA) and allowing formation of a colored precipitate. Immunocytochemistry. Reactivity of these antibodies with isolated rat alveolar type II cells was tested by immunocytochemical analysis. Isolated alveolar type II cells were cultured on glass coverslips and treated with A2C, A2R, or control preparations. Antibody binding was visualized by applying the ABC technique [16]. Diaminobenzidine and H 2O 2 were added to elicit a colored reaction product and slides were examined using an Olympus photomicroscope.
RESULTS
Antibodies Bind to Type II Cells Previous work [10, 11] showed that A2C and A2R, two independently raised aiMAbs, bound a É32-kDa protein in lung homogenates and that the protein they recognized was present at the cell membrane of human, rabbit, and pig alveolar type II cells. However, since functional studies of surfactant secretion are performed with rat alveolar type II cells it is important to ascertain that these rat aiMAbs bind to rat alveolar type II cells. The alveolar type II cell isolation procedure used here, provides preparations that are ú95% type II cells, as determined by fluorescence staining with phosphine 3R [14, 15]. To test whether these type II cells compare to human and other species’ type II cells, which are recognized by A2R and A2C, immunocytochemical staining showed that both A2C and A2R bind to isolated type II cells, at the cell membrane (Fig. 1). We had previously applied Western and ligand blot analyses to determine that A2C and A2R bind a 32-kDa type II cell membrane protein from human and porcine lungs and that this 32-kDa protein also binds SP-A. Therefore, Western blot and ligand blot analyses were used to analyze isolated cell membrane proteins from these enriched rat type II cell preparations. This analysis showed that A2C and A2R bind a single 32-kDa rat alveolar type II cell membrane protein and that a protein of the same size also binds SP-A (Fig. 2). Antibodies Bind to a Receptor and Competitively Inhibit SP-A Binding To ascertain that these type II cell preparations are comparable to those reported by others to study SP-A receptor activity, we measured SP-A receptor number and affinity in our cell preparations. The SP-A binding characteristics of these isolated type II cells were studied by Scatchard analysis. Binding of SP-A to these type II cells was determined (Fig. 3a). We found that type II cell SP-A binding was maximal at 2 mg/ml SPA. Alveolar type II cells were incubated with various
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concentrations of 125I-SP-A, and the free and bound pools were measured [7]. We observed a monophasic SP-A binding curve (Figs. 3a and 3b), indicating that the SP-A receptors on our type II cell preparations bind SP-A with uniform affinity (Kd Å 1.20 { 0.13 mg/ml) and that there are 105 SP-A binding molecules for each type II cell. The shape of the binding curve, indicating positive cooperativity, and the Kd and receptor numbers are virtually identical to those reported by Kuroki et al. [7], indicating that our type II cell preparations are comparable to those reported by others. To characterize further the reactivity of A2C and A2R with type II cell membrane proteins, the inhibition of SP-A binding by these aiMAbs was examined. Iodinated SP-A (5 mg/ml) and various concentrations (between 0 and 10 mg/ml) of unlabeled SP-A, A2C, and A2R were added as potential competitive inhibitors of the SPAR – 125I-SP-A interaction. These inhibitors were incubated with the alveolar type II cells for 4 h. Both antibodies reduce binding of iodinated SP-A to isolated alveolar type II cells (Fig. 3c). The reduction in binding of 125I-SP-A to alveolar type II cells was dose-dependent. At high concentrations of inhibitor, the percentages of inhibition were comparable for A2R, A2C, and unlabeled competing SP-A. Furthermore, maximal reduction of 125I-SP-A binding was seen with 1 mg/ml of A2C, 0.5 mg/ml of A2R, and 10 mg/ml of SP-A.
dependent fashion (P õ 1004 by x2 analysis). Maximal reversal of inhibition was observed at A2C concentrations ú 50 mg/ml, but statistically highly significant increases in secretion occurred at A2C concentrations §1 mg/ml. Normal rat IgG had no effect on SP-A inhibition of phospholipid secretion. These results are consistent with the conclusion that A2C (and A2R) binds to an SP-A receptor and blocks its inhibitory function. The dose–response curves shown in Fig. 4 were produced using isolated alveolar type II cells treated with ATP. A number of compounds are known to stimulate surfactant secretion [2]. SP-A inhibits the action of these secretagogues [8]. However, these secretagogues act through different mechanisms. Thus, it is important to determine whether A2C and A2R also reverse the SP-A induced inhibition of surfactant secretion elicited by other secretagogues. The secretagogues tested (ATP, dibutyryl cAMP, terbutaline, and ionomycin) were selected to represent the variety of modulators known to stimulate surfactant secretion. Regardless of the secretagogue used, alveolar type II cells incubated with 100 ng exogenous SP-A secreted less phospholipid than did cells incubated without SP-A (Fig. 5). A2C reversed the inhibition of secretion by SP-A regardless of the secretagogue, although in some cases A2C did not restore surfactant secretion completely (Fig. 5).
A2R and A2C Reverse SP-A Inhibition of Type II Cell Phospholipid Secretion
DISCUSSION
Anti-idiotype MAbs A2C and A2R bind a É32-kDa type II cell membrane protein and inhibit the binding of SP-A to isolated alveolar type II cells. The function of this SP-A binding protein remained to be determined. Thus, we tested whether this É32-kDa protein also performed the recognized SP-A receptor function for type II cells: regulation of surfactant secretion. When isolated alveolar type II cells are incubated with SP-A, they do not secrete as much phospholipid in response to various secretagogues (e.g., ATP) as do cells incubated without SP-A [8]. To determine whether the É32-kDa protein bound by A2C and A2R is a functional receptor, we preincubated alveolar type II cells with [methyl3 H]choline to label cellular lipids and then treated them with antibody or normal IgG, followed by SP-A (100 ng/ml) and ATP (1 mM). This dose of SP-A blocked type II cell phospholipid secretion. Antibodies A2C (Fig. 4) and A2R (not shown) reversed this inhibition in a dose-
Surfactant secretion by alveolar type II cells is regulated by complex mechanisms that respond to a wide variety of stimuli. Both physical (e.g., deep inflation) and chemical (e.g., purines, adrenergic agonists, and calcium ionophores) stimuli reportedly affect surfactant secretion (reviewed, ref. 2). The means by which physical stimuli increase surfactant secretion are unknown. The cytokines and biologically active chemicals that are surfactant secretagogues may act through at least three different intracellular signaling systems (e.g., increasing Ca2/ and cAMP and activating PKC). The mechanisms by which these various secretagogues modulate surfactant secretion are being elucidated and are still unclear. However, very little is known about the regulation of surfactant secretion by SP-A. Surfactant secretion is regulated by a negative feedback loop in which SP-A, a protein produced by alveolar type II cells, binds to a specific receptor on type II cell mem-
FIG. 1. A2C and A2R antibodies recognize a rat type II cell membrane protein. Type II alveolar pneumocytes were isolated according to [14] and allowed to adhere as usual. The cultures were fixed with acetone, then treated with affinity-purified rat monoclonal anti-idiotype antibody A2R (a, 11100), A2C (b, 11100), or normal rat IgG (c, 1600) (original magnifications). Antibody binding was visualized by the ABC technique (see Materials and Methods). Slides were counterstained with hematoxylin. The aiMAb bound at the plasma membrane of isolated rat type II cells.
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FIG. 2. SP-A and anti-idiotype monoclonal antibodies A2R and A2C bind a 32-kDa rat type II cell membrane protein. Type II alveolar pneumocytes were prepared as usual. Cell membranes were isolated from these cells according to standard procedures (see Materials and Methods), solubilized, separated by SDS– PAGE, and electroblotted to PVDF membranes. These membranes were then exposed to affinity-purified A2C or A2R, followed by rabbit anti-rat IgG then 125I-Staph. protein A. Alternatively, the membranes were exposed to 125I-labeled SP-A (10). The locations of coelectrophoresed molecular size markers are shown at the right. SP-A, A2C, and A2R all bound a 32-kDa protein from rat type II cell membranes.
branes [7–11]. This interaction reduces the rate of surfactant secretion. The identification and characterization of the SP-A receptor has proven difficult. However, we developed two monoclonal aiMAbs for this purpose. The aiMAbs, A2C and A2R, were derived independently by immunizing different rats with different combinations of monoclonal antibodies against surfactant protein. Hybridomas were selected for their ability to block the ability of antibodies against surfactant proteins to bind surfactant protein-A. A2C and A2R bound a É30-kDa non-Ig protein on rabbit, pig, and human lung cell membranes [11]. We have reported the use of these aiMAbs to identify human and porcine SP-A binding proteins in lung cDNA expression libraries and described the structure of these proteins and their cDNAs [10]. The use of aiMAbs to identify an unknown or putative receptor protein is well established. The theoretical basis for this approach was described by Jerne [17]. Used in situations in which a ligand is available and antibodies to the ligand are on hand, the practical application of anti-idiotype antibody approaches for the identification of cell membrane receptors has been amply demonstrated [18–20]. Most recently, this technique has received molecular substantiation with an X-ray crystallographic demonstration that receptor – anti-idiotype interaction may closely resemble, spatially, the receptor–ligand interaction, even when the anti-idiotype and the ligand appear to be much different from each other [21]. We report here that the aiMAbs A2C and A2R bind a É32-kDa non-Ig protein on rat alveolar type II cells.
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This finding is important for several reasons. Our previous reports that A2R and A2C recognized a É30-kDa SP-A binding protein were based on Western blot analysis of whole lung homogenates, immunohistochemical localization in tissue sections, and both Western and ligand blot studies of recombinant human and porcine SP-A binding proteins. The present report uses isolated type II cells to establish the cell-type specificity and the subcellular localization of the SP-A binding protein, i.e., alveolar type II cell membranes. The present work confirms the molecular size of this protein of É32-kDa, which is consistent with the deduced open reading frame sizes for both human and pig SP-A binding protein [10]. The demonstrable reactivity of A2R and A2C with type II cells from the same animal species that produced these aiMAbs (rat) further confirms that type II cells are the targets to which A2C and A2R bind and that the É32-kDa protein recognized by A2C and A2R is a type II cell membrane protein. The data presented here establish the role of this É32-kDa SP-A binding protein as a functional SP-A receptor (SPAR). A2C and A2R interrupt the negative feedback loop that is used by alveolar type II cells to regulate surfactant secretion. Surfactant secretion is stimulated by a number of secretagogues and SP-A inhibits surfactant secretion regardless of the secretagogue used [8]. Thus, the regulatory activity of any putative SP-A receptor should be demonstrably impervious to the variety of secretagogues used. The É32kDa protein recognized by A2C and A2R fits this requirement: A2C and A2R both restore the ability of alveolar type II cells to secrete surfactant in the presence of inhibiting amounts of SP-A regardless of the secretagogue used (ATP, dibutyryl-cAMP, terbutaline, or ionomycin). In their analysis of SP-A receptor avidity and cell membrane density, Kuroki et al. reported additional studies and applied corrections in their calculations, to compensate for SP-A internalization during the study period [7]. Our studies, which were performed in the presence of excess labeled SP-A, were intended to ascertain that our type II cell preparations were comparable to those studied by others in terms of SP-A binding, receptor number, and receptor affinity. Our quantitative binding studies showed that 105 SP-A molecules bound each type II cell and that the affinity of this binding was 1.2 { 0.13 mg/ml. These numbers are comparable to previously reported data (1.35 1 10 5 SP-A molecules bound/cell, Kd Å 1.02 { 0.32 mg/ml [7]). The Scatchard plot of these data is very similar to that reported by Kuroki et al. [7] and shows positive cooperativity in binding. We found that A2R and A2C inhibited SP-A binding to this receptor with similar affinity. These data confirm that A2C and A2R bound the same molecule that binds SP-A and indicate that the ability of these ai-
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FIG. 3. A2C and A2R inhibit SP-A binding to rat alveolar type II cells. Type II alveolar pneumocytes were isolated as according to [14] and cultured overnight to allow attachment to plastic tissue culture plates. The following day, the plates were washed and then treated with fresh medium. (a) Binding of 125 I-SP-A to type II cells was measured as a function of the 125I-SP-A concentration. For Scatchard analysis (b) various concentrations of 125I-SP-A were added, while for analysis of inhibition (c) a constant concentration (5 mg/ml) of 125I SPA and various amounts of unlabeled SP-A, A2C, or A2R. The cells were incubated for 4 h, and the pools of free and bound 125I SP-A were measured as described by Kuroki et al. [7]. Results shown are the means ({SEM) of at least two independent determinations, each with §2 replicates. 125I SP-A binding is similar to that described by Kuroki et al. Furthermore, both A2C and A2R inhibit the binding of 125I SPA in a dose-dependent fashion. Differences between the conditions of 0.01 mg/ml inhibitor on the one hand and all of the antibody concentrations tested on the other; and between SP-A as an inhibitor on the one hand and the antibodies on the other, at concentrations between 0.01 and 1.0 mg/ml are statistically significant (P õ 0.05).
MAbs to interrupt the SP-A –surfactant secretion negative feedback loop may reflect, in part, their ability to displace SP-A from SPAR. A2R and A2C displace 125ISP-A from SPAR more efficiently than does unlabeled SP-A at low concentrations, but comparably to unlabeled SP-A at high concentrations in the same assay. The data presented here, taken together with our previously published reports, support the conclusion that the É32-kDa protein recognized by A2C and A2R is the functional receptor for alveolar type II cells. SP-
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A inhibits surfactant secretion. A2C and A2R, but not nonspecific IgG, reverse this inhibition. A2C and A2R bind one alveolar type II cell membrane protein. Scatchard analysis shows that SP-A binding activity is present on type II cells and that A2C, A2R, and SP-A all bind this receptor in similar fashion. The mechanism by which this SP-A receptor affects surfactant secretion is not understood. One possibility is that even though different secretagogues may generate different intracellular signals and act through
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FIG. 4. A2C antibody restores surfactant secretion by rat type II cells. Type II alveolar pneumocytes were isolated according to [14] and cultured overnight with [methyl-3H]choline as usual. The following day, cultures were treated with ATP, followed by different concentrations of A2C or normal rat IgG, and 100 ng/ml purified bovine SP-A. Surfactant secretion is measured as the percentage of total phospholipid that is secreted within 2 h. Results shown are the means ({SEM) of at least three different determinations. The range (mean { SEM) of surfactant secretion by isolated type II cells not treated or treated only with ATP is shown. Surfactant secretion by isolated type II cells treated with ATP and SP-A was 0.62 { 0.09. The A2C and IgG dose-response curves shown are significantly different from each other by x2 analysis (P ! 0.01). Normally, SP-A inhibits surfactant secretion. A2C, but not normal IgG, restored surfactant secretion by type II cells in a dose-dependent fashion.
mechanisms that are initially different, these diverse signals activate a single pathway of surfactant secretion. SPAR regulates the activity of this distal pathway regardless of the means used to enhance secretion.
SPAR cDNA bears some homology to other (e.g., ryanodine) receptor proteins, in particular cDNAs encoding receptors that are linked to a Ca 2/ second messenger system [10]. Therefore, it is possible SPAR activa-
FIG. 5. Effects of SP-A and A2C on surfactant secretion by rat type II cells stimulated by different secretagogues. Type II pneumocytes were isolated and cultured overnight with [methyl- 3H]choline to label phospholipids as usual. The following day, cultures were treated with one of several different secretagogues: ATP, terbutaline, cAMP, or ionomycin, {5 mg/ml A2C, {100 ng/ml SP-A. Surfactant secretion was measured after 2 h. Results shown are the means ({SEM) of at least three different determinations. SP-A inhibits the surfactant secretion that is stimulated by each of the different secretagogues tested. In all cases, A2C reversed this inhibition and restored surfactant secretion. * P õ 0.02 compared to parallel cultures receiving secretagogue only and compared to parallel cultures receiving secretagogue / SP-A / A2C. ** P õ 0.02 compared to parallel cultures treated with ATP, but without A2C or SP-A. All other groups are statistically indistinguishable from the secretagogue alone group for each individual secretagogue, respectively.
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tion regulates cellular Ca2/ traffic. The involvement of Ca2/ in regulation of surfactant secretion has been suggested previously. A number of surfactant secretagogues (i.e., ATP, ionomycin, and terbutaline) cause transient increases in intracellular Ca2/ of alveolar type II cells [22]. Furthermore, surfactant secretion can be inhibited by incubating alveolar type II cells in the presence of W-7, a known calmodulin blocker [23]. Binding of SP-A to SPAR may potentially inhibit these Ca2/ transients because alveolar type II cells incubated in the presence of SP-A / ionomycin had lower intracellular Ca2/ concentrations than did cells treated with ionomycin alone [24]. In the present experiments SPA inhibited ionomycin-stimulated surfactant secretion, and A2C reversed the SP-A induced inhibition, suggesting that SPAR may modulate surfactant secretion by regulating cytosol calcium. The present studies indicate that these antibodies may potentially be used to treat diseases of surfactant insufficiency, including neonatal and adult respiratory distress syndromes (RDS). Treatment for these diseases currently involves administration of surfactants extracted from animal lungs or prepared artificially. While quite useful for neonatal RDS, surfactant replacement therapy is only being applied experimentally to adult RDS and is quite costly. These antibodies may offer an additional, perhaps supplemental, approach to stimulate secretion of endogenous surfactant. The work presented heretofore would not have been possible without the kind technical help of Ricardo Moraes, Ai-Min Wu, and Meng Zhao. In addition, the support and advice of the following investigators were helpful in our studies: Sandra Bates, Michael Beers, Aron Fisher, Simon Slater, Andrew Thomas, Dennis Voelker, and Jeff Whitsett. Immunocytochemical studies were kindly performed by Al Kovatich and Mike Noble, under the direction of Dr. Roland Schwarting. This work was supported by Grant 2749A from the Council for Tobacco Research, U.S.A., as well as by NIH Grants AA7463 and HL49959.
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Received February 12, 1996 Revised version received April 18, 1996
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AP: Exp Cell