Lamellar body membrane turnover is stimulated by secretagogues

Sandra R. Bates, Jian-Qin Tao, Susanne Schaller, Aron B. Fisher, and Henry Shuman

Institute for Environmental Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Lamellar bodies are specialized cellular organelles used for storage of surfactant by alveolar type II cells of the lung. We utilized monoclonal antibody (MAb) 3C9, which recognizes an integral lamellar body-limiting membrane protein of 180 kDa, to follow lamellar body trafficking. 125I-labeled MAb 3C9 bound to the surface of type II cells and was internalized by the cells in a time- and concentration-dependent manner that was inhibitable by excess unlabeled antibody. The internalized antibody remained undegraded over a 4-h time period. The L2 rat lung cell line that does not have lamellar bodies did not bind iodinated 3C9. Exposure of type II cells to the secretagogues ATP, phorbol 12-myristate 13-acetate, and cAMP resulted in a 1.5- to 2-fold enhancement of binding and uptake of MAb 3C9. Calphostin C inhibited phorbol 12-myristate 13-acetate-stimulated phospholipid secretion and also reduced binding and uptake of MAb 3C9 by type II cells. Treatment of type II cells with phenylarsine oxide to obstruct clathrin-mediated endocytosis had no effect on the internalization of MAb 3C9 while markedly blocking the uptake of surfactant protein A and transferrin. An actin-mediated process was important for lamellar body membrane uptake because incubation with cytochalasin D partially inhibited MAb 3C9 incorporation by type II cells. These studies are compatible with enhanced lamellar body membrane turnover associated with surfactant secretion and indicate that this process can be monitored by the trafficking of the antigen reporter MAb 3C9.

surfactant; type II cells; rat lung; membrane turnover; phosphatidylcholine


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

LUNG SURFACTANT is a complex mixture of proteins and lipids, primarily phospholipid, which is enriched in dipalmitoylphosphatidylcholine and phosphatidylglycerol. Surfactant functions to lower surface tension at the air-water interface of the alveoli in the lung, thereby preventing lung collapse and facilitating oxygen exchange. Alveolar type II cells contain lamellar bodies, the functionally specialized cellular organelles that store surfactant as multilamellar whorls. In addition to surfactant, various proteins are associated with lamellar bodies, including low-molecular-weight GTP-binding proteins (33), lysosomal hydrolases (20), phospholipases (19), and lysozyme (37). Lamellar bodies are also characterized by their acidic internal pH (6) and their high calcium content (11). Lung pneumocytes release surfactant from the lamellar bodies into the alveolar space, where the phospholipid and surfactant proteins form tubular myelin structures in the subphase and monolayers at the air-water interface (41). Lamellar bodies also participate in the recycling of surfactant because lipid and surfactant protein A (SP-A) uptake studies (9, 13, 30, 43) have demonstrated that exogenous phospholipid, as either surfactant or liposomes, and SP-A are taken up by the lung and accumulate in lamellar bodies. Thus lamellar bodies function as both secretory organelles and components of the endocytic pathway.

Although the limiting membranes of lamellar bodies are known to contain H+-ATPase (5); Rab7, usually found in late endosomes (40); LAMP-1, a lysosomal protein (40); and acid phosphatase (24), protein markers specific for the limiting membrane of lamellar bodies had not been described until the recent report by Zen et al. (44). They produced monoclonal antibody (MAb) 3C9, which recognizes an integral lamellar body-limiting membrane protein of 180 kDa (lbm180). In rat lung tissue, MAb 3C9 specifically labeled cuboidal type II cells, and in cultures of isolated type II cells, the label appeared to be localized to the outer ring but not to the interior vacuolar compartment of lamellar bodies. With immunogold labeling of 3C9, gold particles were primarily on the lamellar body membrane, with label also seen on multivesicular bodies and small cytoplasmic vesicles. An immunofluorescent study (44) demonstrated that the presence of lbm180 protein correlated temporally with the appearance of lamellar bodies in fetal rat lung preparations and the disappearance of lamellar bodies during long-term culture of isolated type II cells as determined by phase-contrast microscopy.

The synthesis, secretion, and internalization of surfactant phospholipids and proteins by type II cells have been studied extensively. However, movement of lamellar bodies between intracellular compartments and the plasma membrane is poorly understood. Recent work (17) with fluorescent dyes visualized the fusion of lamellar bodies with the plasma membrane and documented single exocytotic events. In the present study, iodinated MAb 3C9 was used to follow the appearance of lamellar body-specific lbm180 protein on the surface of type II cells and its retrieval into the cells under various conditions to characterize lamellar body membrane trafficking. The results provide evidence that 1) secretagogue treatment enhances the turnover of lamellar bodies and 2) lamellar body membrane retrieval is partially actin dependent.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cell Preparation

Type II cells were isolated from the lungs of anesthetized male Sprague-Dawley rats according to the method of Dobbs et al. (10) as described previously (1). Briefly, perfused, lavaged lungs were digested with elastase and minced with a McIlwain tissue chopper. The isolated cells were sequentially filtered through nylon meshes (160, 37, and 10 µm) and enriched for type II cells by plating on rat IgG (Sigma, St. Louis, MO)-coated petri dishes for 1 h at 37°C to remove macrophages. After overnight culture and removal of nonadhered cells, the purity of the type II cell preparation was routinely >93% by modified Papanicolaou stain.

Type II cells were plated on 35-mm plastic tissue culture dishes (Costar, Cambridge, MA) at 3 × 106 cells/dish or on 24-mm inserts of Transwell microporous membranes (3-µm pore size; Costar) at 5 × 106 cells/well. Cells were cultured in 10% FCS in MEM at 37°C in a humidified incubator with 5% CO2 in air. The L2 cell line derived from rat lung was obtained from the American Type Culture Collection (Manassas, VA) and grown in MEM supplemented with 10% FCS. Alveolar macrophages were isolated by centrifugation of rat lung lavage fluid and placed in culture with MEM containing 10% FCS for 24 h before use. The purity of the resultant macrophage preparation was >98%. Cell protein was measured by the Lowry (27) or Bradford (4) method.

Purification of SP-A

Bronchoalveolar lavage fluid of lungs from alveolar proteinosis patients obtained after therapeutic lavage at the Hospital of the University of Pennsylvania (Philadelphia) was centrifuged to remove cellular material. Surfactant was purified with density gradient centrifugation followed by dialysis and lyophilization as described previously (13). SP-A was isolated from surfactant according to the method of Hawgood et al. (18) with 1-butanol and beta -D-glucopyranoside extraction, dialysis, and microconcentration. As described previously (1), the purity of the SP-A preparation was monitored with SDS-PAGE according to the method of Laemmli (26).

Monoclonal Antibodies and Immunofluorescence

The preparation of MAb 3C9 from BALB/c mice immunized with the membrane fraction of rat lung lamellar bodies was described previously (44). MAb 3C9 was purified by precipitation from the hybridoma supernatant with (NH4)2SO4 and stored in aliquots. In the present study, immunofluorescence was performed on type II cells placed in culture for 24 h, fixed and permeabilized with cold methanol-acetone, incubated with purified MAb 3C9 for 2 h, and incubated with Texas Red-conjugated goat anti-mouse IgG as described (44). The cells were observed with an inverted Nikon fluorescence microscope.

Iodination

Antibodies, transferrin, or SP-A 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 for 3C9, nonimmune IgG, transferrin, and SP-A was 90 ± 58 (n = 13 determinations), 492 ± 54 (n = 3 determinations), 351 ± 139 (n = 3 determinations), and 495 ± 110 (n = 9 determinations) counts · min-1 (cpm) · ng protein-1, respectively. The percentage of TCA precipitability for 3C9, nonimmune IgG, transferrin, and SP-A was 91.4 ± 1.1 (n = 13 determinations), 96.5 ± 0.5 (n = 3 determinations), 94.8 ± 1.1 (n = 3 determinations), and 92.8 ± 0.2% (n = 9 determinations), respectively. The iodinated proteins were stored at 4°C and used within 3 wk. Storage of MAb 3C9 did not appreciably affect the TCA precipitability of the antibody.

Incubation of Iodinated Ligands or Antibodies With Type II Cells

Type II cells plated on plastic dishes or on Transwell membranes were cultured for 20-24 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-3C9 with type II cells, cells were incubated with 1 µg/ml of 125I-3C9 and 1 mg/ml of BSA in the presence of 1.0 µg/ml of nonlabeled 3C9 or IgG at 37°C for 1 h. For binding and uptake studies, type II cells were incubated with and without secretagogues for 30 min at 37°C, followed by the addition of iodinated ligands for the indicated time period. To terminate both types of experiments, the nonbound 3C9 was removed by three washes with MEM and two washes with PBS at 4°C. The cells were harvested with trypsin and washed twice by centrifugation in PBS. Bound ligand was defined as trypsin-sensitive material, whereas internalized ligand was trypsin resistant. For binding studies at 4°C, the cells were washed twice with cold MEM, incubated at 4°C for 1 h with increasing concentrations of MAb 3C9, washed with cold MEM (3 times) and PBS (2 times), and dissolved in 0.2 N NaOH. Aliquots were taken for protein and radioactive determination. The amount of 125I-3C9 associated with the cells was measured with a gamma counter. Background binding by dishes or wells without cells was determined in every experiment, and the amount of radioactivity in the absence of cells was subtracted from the samples. The results were normalized to cellular protein content.

Cellular Viability

Phenylarsine oxide. Phenylarsine oxide (PAO), a trivalent arsenical, has been used to block clathrin-mediated endocytosis (28). At concentrations >10 µM, PAO is known to inhibit cellular oxygen consumption and reduce ATP levels (14). Thus rhodamine 123 (R-123), a cationic fluorescent probe that accumulates in negatively charged organelles, especially mitochondria, was used to monitor cell viability during exposure to PAO (22). Rhodamine is also sequestered in lamellar bodies due to their internal acidic pH (6). A reduction in R-123 fluorescence from the cell and appearance of the fluorophore in the medium have been associated with mitochondrial membrane depolarization and loss of cell viability (16). In type II cells prelabeled with R-123 and exposed to nitric oxide or peroxynitrite, there was a marked loss of intracellular R-123 fluorescence together with other evidence of cell death (16). Type II cells were incubated with 10 µM R-123 for 1 h, the cells were washed, and PAO was added at 2 µM for various times. Rhodamine fluorescence intensity was measured in the medium in arbitrary fluorescence units at an excitation of 500 nm and emission of 536 nm (16).

Lactate dehydrogenase release. To further evaluate the possibility of toxic effects of PAO, lactate dehydrogenase (LDH) activity was measured in the supernatant of cells in the absence and presence of 2 µM PAO. After exposure to PAO for 20 min, the medium was centrifuged, and the supernatant and the cells on the dish were assayed for LDH activity and compared with activity in control medium and cells.

Phosphatidylcholine Secretion

After isolation, type II cells were incubated overnight with 0.5 µCi of [methyl-3H]choline chloride (Amersham, Arlington Heights, IL) in MEM containing 10% FCS to label cellular phospholipids. Cells were washed six times and incubated for 30 min in MEM to measure phosphatidylcholine (PC) secretion as described previously (2). One set of cells was harvested at time 0 and served as a control for phospholipid secretion associated with the medium change. The remaining cells were incubated with and without additions for another 2 h. The medium was removed and centrifuged to remove detached cells. Methanol was added to the cell monolayer, and the cells were scraped from the dish. The cells and the medium were extracted with the Bligh and Dyer (3) method. The amount of phospholipid secretion was calculated as the percentage of lipid counts per minute in the medium relative to the total counts per minute of lipid present in the cells plus the medium. All experiments were performed in duplicate, and the values were averaged.

Materials

PAO, transferrin, and cytochalasin D were obtained from Sigma.

Statistical Analysis

Results are reported as means ± SE unless otherwise stated. Results were analyzed statistically by t-test or paired t-test with SigmaStat for Windows (Jandel, San Rafael, CA), where significance was taken as P < 0.05. When SE bars are not visible in Figs. 2-7, they are contained within the symbols or bars.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Immunofluorescent Localization of MAb 3C9 to Lamellar Bodies of Type II Cells

As shown in Fig. 1, purified MAb 3C9 immunolabeled the lamellar bodies of type II cells grown in culture for 24 h as demonstrated previously with the MAb 3C9 hybridoma supernatant (44). The MAb 3C9 label was found in bright circles surrounding vacuolar structures (Fig. 1B), which demonstrated a morphology consistent with lamellar bodies in the paired phase-contrast image (Fig. 1A).


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Fig. 1.   Immunofluorescence localization of monoclonal antibody (MAb) 3C9 binding to lamellar bodies of type II cells in culture for 24 h. A: phase-contrast micrograph. B: fluorescent micrograph. MAb 3C9 strongly labels lamellar body membranes that surround lamellar bodies in type II cells. Bar, 10 µm.

Time and Concentration Dependence of the Cell Association of MAb 3C9

The time course for the binding of the 125I-labeled MAb 3C9 to the lamellar body membrane protein on the surface of type II cells and its internalization by type II cells is shown in Fig. 2. Maximum binding of 3C9 to the surface of the cells was complete after 1 h of incubation. Once bound, the antibody was taken up into a trypsin-resistant compartment of the cell (Fig. 2). The internalization continued fairly linearly throughout the course of the experiment. Degradation of the iodinated antibody did not occur based on the lack of an increase in TCA-soluble counts in either the medium or the cells above background over the 4-h time period. As shown in Fig. 3, the cell association of 3C9 to type II cells varied with increasing concentrations of 3C9. During 1 h of incubation at either 4°C (Fig. 3A) or 37°C (Fig. 3B), the binding of MAb 3C9 to type II cells increased linearly up to 1 µg/ml of 3C9, whereupon the slope of the binding curve became less steep, indicating that a limited amount of ligand protein was present on the cell surface. Over the range of 3C9 levels in the medium from 0.25 to 6 µg/ml, the amount of antibody internalization was ~65% of the level of 3C9 bound to the type II cells.


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Fig. 2.   Time course of binding and uptake of MAb 3C9 by type II cells. Type II cells were incubated with MAb 3C9 (1 µg/ml) for indicated times. Cells were harvested with trypsin to separate trypsin-sensitive (binding) and trypsin-resistant (uptake) compartments. Data are means ± SE; n, no. of experiments performed in duplicate.



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Fig. 3.   Characteristics of binding and uptake of MAb 3C9 by type II cells. Increasing concentrations of MAb 3C9 were added to type II cells for 1 h at 4 (A) or 37°C (B). Data are means ± SE; n, no. of experiments, each performed in duplicate. Extent of cell binding at 4°C (A) was calculated as percentage of binding at 1 µg 3C9/ml = 100% (control). Control value was 32 ± 1.5 ng 3C9/mg cell protein. Extent of cell binding and uptake at 37°C (B) were calculated as percentage of uptake of 3C9 at 1 µg 3C9/ml = 100% (control). Control value was 80 ± 16 ng 3C9/mg cell protein.

Specificity of the Interaction of MAb 3C9 and Type II Cells

The interaction of MAb 3C9 with its ligand on the type II cell surface could be competed with excess 3C9. Type II cells were incubated with increasing concentrations of unlabeled 3C9 or nonimmune rat IgG for 10 min. 125I-3C9 (1 µg/ml) was added, and the experiment continued for 1 h at 37°C. Coincubation of iodinated 3C9 with a fivefold excess of unlabeled 3C9 reduced both binding and uptake of labeled antibody by 63% relative to control values (Fig. 4). Thus the competition of unlabeled 3C9 with 125I-3C9 for the binding and uptake by the type II cells was concentration dependent. Unlabeled nonimmune IgG had no effect on the cell association of 125I-3C9.


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Fig. 4.   Competition of MAb 3C9 binding and uptake by excess unlabeled 3C9. Increasing concentrations of unlabeled MAb 3C9 or nonimmune rat IgG were added to type II cells for 10 min, followed by addition of 125I-3C9 (1 µg/ml) and incubation for 1 h at 37°C. Data are means ± SE of percentage of binding or uptake of 125I-3C9 in absence of unlabeled ligand (control); n, no. of experiments performed in duplicate. Control values for binding and uptake were 108 ± 16 and 54 ± 2.7 ng 3C9/mg cell protein, respectively.

L2 cells, a cell line isolated from rat lung, do not contain lamellar bodies and, as shown in Table 1, do not bind or incorporate 3C9. These data provide evidence that MAb 3C9 does not react nonspecifically with rat cells. The cell association of 3C9 to alveolar macrophages was 6-13% of that seen for type II cells (Table 1) and is compatible with Fc receptor binding. The association of 3C9 to either the L2 cell line or macrophages did not change with ATP (1 mM) treatment (data not shown).

                              
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Table 1.   Cell association of MAb 3C9 to various cell types

The association of 3C9 to type II cells was specific for this antibody. At 0.5 µg/ml of iodinated IgG, type II cells bound 33 ± 1.3 ng (n = 3 experiments) or incorporated 67 ± 12 ng 3C9/mg cell protein but bound only 1.3 ± 0.1 ng or incorporated 0.9 ± 0.2 ng nonimmune IgG/mg cell protein after a 2-h incubation period. Thus iodinated 3C9 was bound or incorporated by type II cells 25- to 50-fold more than IgG.

Regulation of the Cell Surface Appearance and Retrieval of the Lamellar Body Membrane Protein

Treatment of type II cells with secretagogues results in an increase in both the release and uptake of surfactant phospholipids and proteins (12). The enhancement of the secretion of surfactant occurs presumably via a mechanism that involves movement of lamellar bodies to the cell surface, fusion with the plasma membrane, release of the lamellar body surfactant contents, and retrieval of the lamellar body membrane components into the cell. Because 3C9 interacts with a lamellar body membrane protein, exposure to secretagogues should increase the binding of 3C9 to the surface of type II cells and stimulate the internalization of 3C9. In time-course studies shown in Fig. 5, type II cells were incubated with iodinated 3C9 alone or together with a combination of three known secretagogues for surfactant, ATP (1 mM), phorbol 12-myristate 13-acetate (PMA; 10 nM), and 8-bromo-cAMP (cAMP; 0.1 mM), and cells were harvested at selected intervals thereafter. Secretagogue treatment significantly enhanced both binding and uptake of 3C9 by type II cells after 30 min of incubation. Two hours of exposure to ATP, PMA, and cAMP stimulated binding and uptake of 3C9 by about 1.5-fold compared with that in control cells.


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Fig. 5.   Time course of secretagogue-mediated stimulation of MAb 3C9 cell association. A: binding. B: uptake. 125I-3C9 (0.5 µg/ml) was added to type II cells in absence (control) or presence of ATP (A; 1 mM), phorbol 12-myristate 13-acetate (P; PMA; 10 nM), and 8-bromo-cAMP (cAMP; C; 0.1 mM) in combination. Data are means ± SE; n, no. of experiments performed in duplicate. Control value was 17.9 ± 2.2 ng 3C9/mg cell protein (n = 3 experiments). * P < 0.05 vs. control.

To examine the effects of each secretagogue individually, either ATP (1 mM), PMA (10 nM), or cAMP (0.1 mM) was added to type II cells. Because 30 min were required to measure a secretagogue effect, the cells were preincubated with the secretagogue for 30 min, followed by addition of 125I-3C9 (0.5 µg/ml) for 2 h. Each secretagogue significantly stimulated the binding of 3C9 to type II cells by 1.3- to 2.1-fold compared with that in control cells (Fig. 6A). The addition of all three secretagogues in combination was not additive because binding of 3C9 to the cells was not enhanced further than the binding produced by ATP or PMA individually. Furthermore, as seen in Fig. 6B, secretagogues significantly enhanced the uptake of 3C9 by type II cells, albeit not to the same extent as binding (Fig. 6A). Again, the combined treatment with all three secretagogues was not significantly more potent than that with each secretagogue alone. For 125I-labeled nonimmune IgG, ATP exposure did not affect the binding (control, 3.3 ± 1.1 ng IgG/mg cell protein; ATP, 2.7 ± 0.8 ng IgG/mg cell protein) or uptake (control, 4.7 ± 0.1 ng IgG/mg cell protein; ATP = 3.3 ± 0.5 ng IgG/mg cell protein) of the IgG (n = 3 experiments).


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Fig. 6.   Effect of secretagogues on cell association of MAb 3C9 to type II cells. A: binding. B: uptake. Type II cells were incubated for 30 min with no additions (control), ATP (1 mM), PMA (10 nM), cAMP (0.1 mM), or a combination of ATP, PMA, and cAMP. 125I-3C9 (0.5 µg/ml) was added for an additional 2 h, and cells were harvested with trypsinization. Values are means ± SE; n, no. of experiments performed in duplicate. Control values for binding and uptake were 40.2 ± 3.3 and 59.7 ± 9.1 ng 3C9/mg cell protein, respectively. * P < 0.05 vs. control.

Because stimulation of phospholipid secretion enhanced the interaction of MAb 3C9 with type II cells, inhibition of secretion should reduce MAb 3C9 binding and uptake. This was shown to be the case in the experiments described in Fig. 7. The PMA-stimulated increase in PC secretion was blocked by 10 µg/ml of calphostin C, a protein kinase C inhibitor (Fig. 7A). In concert with secretion, the enhanced binding (Fig. 7B) and uptake (Fig. 7C) of MAb 3C9 produced by incubation with PMA was prevented by inclusion of calphostin C in the medium. Thus the extent of cell association of MAb 3C9 responded similarly to the levels of PC secretion.


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Fig. 7.   Calphostin C (Cal C) inhibits PMA-stimulated phospholipid secretion and cell association of MAb 3C9. A: freshly isolated type II cells were incubated with [3H]choline overnight, washed, and incubated without additions (control) or with Cal C (0.5 µM), PMA (10 nM), or PMA + Cal C. Phospholipid secretion was determined over a 2-h period as described in METHODS. Data are means ± SE of phosphatidylcholine (PC) secretion; n, no. of experiments. Control value was 2.7 ± 0.7%. B and C: binding and uptake, respectively, of 125I-3C9 by type II cells. Type II cells were incubated with additives as in A for 15 min followed by addition of iodinated 3C9 (0.5 µg/ml) for an additional 2 h. Data are means ± SE; n, no. of experiments. Control values for binding and uptake were 22.2 ± 5.7 and 46.7 ± 9.2 ng 3C9/mg cell protein, respectively. Significantly different (P < 0.05) from: * control value; #PMA value.

Pathways of Internalization of 3C9

PAO is a trivalent arsenical that blocks receptor-mediated endocytosis, probably by cross-linking clathrin. It has been shown to inhibit clathrin-mediated uptake of ligands (14, 21, 23) without affecting ligand binding (28). To examine the role of clathrin in the internalization of 3C9, we used two proteins as models: transferrin, a ligand for which internalization is known to occur via clathrin-coated pits, and SP-A, a protein that binds to a specific cell surface receptor on type II cells (8, 24, 42) and has been localized in coated pits (34). Concentrations of PAO > 10 µM are known to affect cellular energy stores (14). To determine the viability of type II cells incubated with 2 µM PAO, rhodamine fluorescence released into the medium by cells previously loaded with R-123 was followed (16). Rhodamine was released into the medium at a slow rate from both the control and treated cells over the experimental period, probably due to its location in secreting lamellar bodies. However, the amount of R-123 in the medium of cells incubated in 2 µM PAO did not differ from that in the medium of control cells over a 40-min incubation period, and after an additional 20 min of incubation time with 10 µM PAO (total elapsed time of 60 min), PAO-exposed cells showed only a 10% enhancement of rhodamine release over control values (data not shown). After 20 min of incubation in 2 µM PAO, rhodamine fluorescence in the medium was 104 ± 5% of that in control cells (arbitrary fluorescence units/dish; n = 3 experiments). Figure 8 shows light and fluorescent photomicrographs of type II cells labeled with R-123 and incubated with 2 µM PAO. In addition to mitochondria, R-123 labeled phase-dense intracellular organelles with the morphological characteristics of lamellar bodies (Fig. 8, A and B), which are known to be acidic (6). After 20 min of exposure to PAO, the cells retained normal morphology and R-123 fluorescence (Fig. 8, C and D). Release of LDH was used as a further determination of cellular viability. LDH released into the medium as a percentage of the total LDH in the tissue culture dish (cells plus medium) in the presence of 2 µM PAO for 20 min (5.6 ± 0.8%) was not significantly different from that in control cells (3.9 ± 0.6%; n = 3 experiments). Thus there was no biochemical or morphological indication of cellular toxicity after 20 min of exposure to 2 µM PAO.


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Fig. 8.   Phase (A and C) and fluorescent (B and D) microscopy of type II cells labeled with rhodamine 123 and incubated with 2 µM phenylarsine oxide (PAO). Type II cells were incubated with 10 µM rhodamine 123 for 1 h. Cells were washed and exposed to 2 µM PAO for 1 (A and B) or 20 min (C and D). Rhodamine 123 fluorescence in mitochondria was not apparent due to brightness of lamellar bodies. Bar, 10 µm.

Exposure of pneumocytes to 2 µM PAO for 20 min had no effect on the binding of 125I-labeled transferrin, 125I-SP-A, or 125I-3C9 (Fig. 9). However, PAO treatment reduced the uptake of transferrin and SP-A, thus supporting the observations of others (34) that SP-A endocytosis occurred via clathrin-coated pits. PAO exposure had little effect on the uptake of 3C9 by type II cells, which indicates that clathrin-mediated pathways are not involved in lamellar body membrane retrieval (Fig. 9).


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Fig. 9.   Effect of PAO on binding and uptake of ligands by type II cells. Type II cells were exposed to PAO for 20 min with either 125I-transferrin, 125I-SP-A, or 125I-3C9 at 0.5 µg/ml and harvested. Values are means ± SE; n = 3 experiments performed in duplicate. PAO does not affect uptake of MAb 3C9 but inhibits uptake of other 2 ligands. * P < 0.05 vs. control cells.

A second uptake pathway involves the budding off of membrane vesicles promoted by actin microfilaments (15). Actin filaments have been observed in close approximation to lamellar bodies, and the depolymerization of the actin network in type II cells with cytochalasin D has been shown to abolish agonist-stimulated phospholipid secretion (39) and reduce liposome uptake (30). As shown in Table 2, a 1.5-h incubation of type II cells with cytochalasin D (10 µg/ml) resulted in a 40% inhibition of the uptake of 3C9 compared with that in control cells while having no effect on the uptake of SP-A or transferrin. Exposure to cytochalsin D did not affect the binding of any of these ligands to the pneumocytes (data not shown), nor did it significantly affect LDH release from the treated cells (7.0 ± 0.7%) compared with that from control cells (5.2 ± 0.8%; n = 3 experiments). Thus some portion of the internalization of the 3C9 ligand protein occurred via an actin-dependent pathway.

                              
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Table 2.   Effect of cytochalasin D on uptake of ligands by type II cells


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The release of surfactant from type II cells occurs through the fusion of lamellar body membranes with the plasma membrane of the cell (17, 35). In this report, iodinated MAb 3C9 raised against lamellar body membranes (44) was used to monitor the appearance of lamellar body membrane protein lbm180 on the surface of type II cells. Morphological studies with MAb 3C9 verified antibody labeling of type II epithelial cells in rat lung cryosections (44) and of lamellar body membranes from cultured type II cells (44 and present study). The data are consistent with the conclusion that lamellar body fusion with the plasma membrane is followed by a retrieval of the lamellar body membrane proteins back into the cell, and both processes may be regulated concurrently.

One hour of incubation was required for the cell surface binding between MAb 3C9 and lbm180 protein to plateau. There were two variables that contributed to the time course of binding of 3C9 to the cells. One was the time required for equilibration of the association between MAb 3C9 and lbm180 protein. Because phospholipid secretion was occurring during the measurement of the binding of 3C9 to type II cells, the second variable was the normal turnover of lbm180 protein at the surface of the cell due to the fusion of lamellar body membranes with the plasma membrane in association with secretion events, followed by the retrieval of lbm180 protein into the cell. The binding data indicate that both variables reached a steady state after 1 h of incubation. Because the results showed that MAb 3C9, and thus lbm180 protein, continued to enter the cell during the experiment, new ligands, i.e., different lamellar bodies, must have moved to the cell surface during the incubation period to keep the amount of lbm180 protein on the cell surface constant. Without a continual replenishment of the plasma membrane with lamellar body membranes, the amount of iodinated MAb 3C9 found on the cell surface would have increased due to binding to the surface lbm180 protein and then progressively decreased with time of incubation as the lbm180 protein was internalized.

When measured with fluorescent dyes, agonist-induced fusion of lamellar body membranes with the plasma membrane occurs within seconds or minutes and terminates within 1 h (17). However, measurement of the release of phospholipid from the cells into the medium with labeled phospholipid indicates that secretion continues for at least 2 h (7). The cell association of MAb 3C9 provides a third method to follow the secretion events. We were not able to see a secretagogue-mediated enhancement of 3C9 cell association with the cells until 30 min after exposure to agonist. This is probably due to the 30 min of time necessary for MAb 3C9 and lbm180 protein to reach binding equilibrium. However, the extent of binding and uptake of 3C9 remained elevated from 30 min to 2 h after exposure to secretagogues, suggesting that the rate of lamellar body fusion with plasma membrane (indicated by the binding of 3C9 to the cell surface) and retrieval of lbm180 continued at an elevated rate up to 2 h after agonist treatment. Although secretion events measured at 37°C by 3C9 binding continued over a longer period of time than those measured at 25°C with fluorescent dyes (17), this may be due to differences in incubation temperature.

The interaction of MAb 3C9 with the type II cells was specific in that it was saturable with increasing concentrations of MAb 3C9 and could be competed with unlabeled MAb 3C9, whereas nonimmune IgG had no effect. In addition, the transformed rat lung cell line L2, which does not possess lamellar bodies, demonstrated little binding or incorporation of MAb 3C9. Due to the presence of some contaminating macrophages in the type II cell preparation, it was important to determine their interaction with 3C9. The low level of binding and uptake of 3C9 by macrophages was probably due to their known ability to phagocytose antibodies. None of the cell types, other than type II alveolar cells, responded to secretagogue treatment by alteration of their interaction with MAb 3C9.

Once bound to lbm180 protein on the cell surface, MAb 3C9 was internalized into a trypsin-insensitive compartment of the type II cell. Internalization of MAb 3C9 was measurable after 15 min of incubation, indicating that lamellar body membrane proteins turn over fairly rapidly. Changes in the extent of internalization of MAb 3C9 occurred in concert with those of the binding of the antibody over a range of 3C9 concentrations, with secretagogue treatment, and with time in culture. No evidence of the degradation of the MAb was found after 4 h of incubation, suggesting that internalized antibody was not transported to lysosomes. In fact, similar studies using fluorescently labeled MAb 3C9 have shown that MAb 3C9 incorporated into type II cells could be found in lamellar bodies after 10 min of incubation, indicating that the lamellar body protein was recycled from the cell surface back to lamellar bodies (36). The present study showed that secretagogue treatment enhanced both the appearance of lbm180 protein on the surface of the cells as well as the removal of this lamellar body membrane protein into intracellular compartments. Thus the turnover of lbm180 protein, and presumably of lamellar bodies, was augmented by secretagogues.

The extent of the cell association of MAb 3C9 to the type II cell surface was qualitatively similar to the level of secretion of surfactant. The secretagogues ATP, PMA, and cAMP, which have been shown to stimulate the release of labeled phospholipid from type II cells (for reviews, see Refs. 31, 32, 38), also promoted the binding and uptake of MAb 3C9. In addition, the inhibition of agonist-stimulated secretion with calphostin C reduced the interaction of MAb 3C9 with the cells. However, phospholipid secretion and the cell association of MAb 3C9 were not quantitatively related. The amount of agonist-stimulated phospholipid secretion (with PMA, 2.9-fold over that in unstimulated control cells) was greater than that seen for the changes measured for MAb 3C9 binding and uptake (with PMA, 1.5- to 1.7-fold over those in control cells). This disparity is likely to be due to the presence of lbm180 on the surface of the cell at the initiation of the experiment as a result of previous secretion events and the rapid recycling of the lbm180 protein.

The uptake of iodinated MAb 3C9 is not dependent on clathrin-coated pits because treatment with PAO had no effect but is dependent on actin because cytochalasin D exposure inhibited the process. Actin may play a role in lamellar body secretion by aiding the movement of the lamellar body to the cell surface, but removal of the actin cortex also might reverse a barrier to fusion of the lamellar body membrane with the plasma membrane. These two opposing roles of actin together with other unidentified mechanisms may account for the observation that inhibition by cytochalasin D was only partial.

SP-A interacts with type II cells via one or more specific cell surface receptors (8, 25, 42). The internalization of SP-A is felt to be mediated by clathrin because electron microscope studies found immunogold-labeled SP-A in both clathrin-coated pits and coated vesicles (34). The data presented here provide further evidence that SP-A endocytosis requires clathrin because PAO blocked SP-A endocytosis under the same conditions that blocked the internalization of transferrin, a ligand with an uptake that is known to be clathrin mediated. Thus the results suggest that SP-A interacts with type II cells via the classic receptor-mediated endocytotic pathway described for many other ligands (29).

With the use of MAb 3C9, which recognizes a lamellar body-limiting membrane protein of 180 kDa, it is now possible to determine exocytotic events in the type II cell by the measurement of the amount of binding of MAb 3C9 to lbm180 protein. This measurement indicates the presence of lamellar body membrane protein on the surface of the cells due to the fusion of the lamellar body with the plasma membrane. The uptake of MAb 3C9 follows the retrieval of lbm180 protein, which would occur in parallel to the movement of lamellar bodies. The cell association of MAb 3C9 was not quantitatively related to phospholipid secretion but changed in concert with it. MAb 3C9 binding and uptake was enhanced on stimulation of phospholipid secretion with secretagogues and inhibited on inhibition of secretion with calphostin C. Thus MAb 3C9 provides a useful tool to follow the trafficking of the important surfactant secretory organelle of type II cells, the lamellar body.


    ACKNOWLEDGEMENTS

We are grateful to Drs. Michael F. Beers and Paul Lanken for aid in obtaining surfactant from alveolar proteinosis patients and to Kathy Notarfrancesco and the Microscopy Core for assistance in the preparation of photomicrographs. We thank Kristine DeBolt for excellent technical support.


    FOOTNOTES

This research was supported by National Heart, Lung, and Blood Institute Grant HL-19737.

Preliminary results of portions of this work were presented at the 1999 American Thoracic Society International Conference in San Diego, CA.

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

Address for reprint requests and other correspondence: S. R. Bates, Institute for Environmental Medicine, Univ. of Pennsylvania, 36th and Hamilton Walk, 1 John Morgan Bldg., Philadelphia, PA 19104 (E-mail: batekenn{at}mail.med.upenn.edu).

Received 3 March 1999; accepted in final form 15 September 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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