Surface-expressed lamellar body membrane is recycled to lamellar bodies

S. Schaller-Bals, S. R. Bates, K. Notarfrancesco, J. Q. Tao, A. B. Fisher, and H. Shuman

Institute for Environmental Medicine, University of Pennsylvania Medical Center, Philadelphia, Pennsylvania 19104-6068


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Monoclonal antibody (MAb) 3C9, an antibody generated to the lamellar body of rat lung type II pneumocytes, specifically labels the luminal face of the lamellar body membrane. To follow the retrieval of lamellar body membrane from the cell surface in these cells, MAb 3C9 was instilled into rat lungs. In vivo, it was endocytosed by type II cells but not by other lung cells. In type II cells that were isolated from rat lungs by elastase digestion and cultured on plastic for 24 h, MAb 3C9 first bound to the cell surface, then was found in endosomes, vesicular structures, and multivesicular bodies and, finally, clustered on the luminal face of lamellar body membranes. The amount internalized reached a plateau after 1.5 h of incubation and was stimulated with the secretagogue ATP. In double-labeling experiments, internalized MAb 3C9 did not completely colocalize with NBD-PC liposomes or the nonspecific endocytic marker TMA-DPH, suggesting that lamellar body membrane is retrieved back to existing lamellar bodies by a pathway different from that of bulk membrane and may be one pathway for surfactant endocytosis. The lamellar body membrane components are retrieved as subunits that are redistributed among the preexisting lamellar bodies in the cell.

endocytosis; membrane trafficking; organelle biogenesis; type II pneumocytes


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

MEMBRANE-BOUND COMPARTMENTS in eukaryotic cells allow specialized and segregated functions to occur in distinct environments. The trafficking between compartments, including the processes of exocytosis and endocytosis, uses a highly conserved fusion mechanism that is mediated by protein-protein interactions between soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNARE proteins) (27). In the SNARE hypothesis, each kind of transport vesicle has its own unique vesicle-SNARE protein that forms a match with its cognate target-SNARE protein at the targeted membrane (26). SNARE proteins were first isolated from neuronal cell membrane because of the large number of secretory vesicles in neurons. The synaptic vesicle cycle at the nerve terminal is the most tightly regulated and is the best characterized membrane-trafficking process (28). The vesicle- and target-SNARE proteins of the synapse, vesicle-associated membrane protein and syntaxin respectively, are also found in the secretory granule and plasma membranes of a number of other cell types that package and store high concentrations of substances in dense core vesicles for regulated export. In particular, SNARE proteins have been found in adrenal chromaffin cells, pancreatic islet cells, pituitary cells, pancreatic acinar exocrine cells, and mast cells (12, 20). These secretory cells also internalize the surface-expressed secretory granule membranes for reutilization (1). In neurons, the synaptic vesicle membrane is retrieved rapidly from the surface as an intact entity and reutilized for neurotransmitter release without additional sorting (24). The membranes of chromaffin granules are selectively retrieved from the plasma membrane, primarily in coated vesicles, sorted through Golgi/post-Golgi compartments, and partly recycled to newly formed chromaffin granules (25).

The work reported here focuses on the recycling pathway of the secretory granule membrane of an exocrine cell in the lung, the alveolar epithelial type II cell. Type II cells are responsible for the production and secretion of surfactant, a lipid-protein complex that lowers surface tension at the air-water interface in alveoli, thus preventing lung collapse during expiration. Before secretion, surfactant is stored in secretory granules called lamellar bodies (2, 4). Each type II cell normally contains 150 ± 30 lamellar bodies (6). Surfactant is released into the alveolar space after fusion of the secretory granule membrane with the cell surface (27), with an in vivo release rate of ~15 lamellar bodies/h (31). In the alveolar space, surfactant is converted into a surface-active monolayer. Type II cells are not only responsible for the production and secretion of surfactant, they are also involved, interestingly, in the clearance of surfactant from the alveolar space. Our laboratory previously showed that surfactant-like lipids are internalized by both a clathrin- and an actin-mediated pathway (23) and that the endocytosed lipid is either degraded or directly deposited in lamellar bodies (5, 9).

Secretion and internalization of surfactant are strongly coupled in type II cells as suggested by the stimulation of both processes by secretagogues (10), but the mechanism for coupling has not been investigated. We hypothesized that surfactant internalization might be coupled to the retrieval of the surface-expressed lamellar body membrane (31). The reutilization of secretory granule membranes by specialized local endocytosis has been shown in neurons and assumed for chromaffin cells because of the longer half-life of the membrane proteins compared with the half-life of the secretory proteins (21, 29). With the generation of a monoclonal antibody (MAb), 3C9, against a 180-kDa lamellar body membrane protein of type II cells (32), we are now able to directly visualize 1) the cellular compartments to which membranes are retrieved, 2) the time course of endocytosis, and 3) the path taken by the recycled secretory granule membrane of type II cells.

To approach these questions, we labeled MAb 3C9 with fluorescent tags (rhodamine and fluorescein) or 10-nm colloidal gold and followed its internalization in whole lungs in vivo and in type II cells isolated from lungs (8) and cultured on plastic for 24 h. To identify the endocytic compartments of lamellar body membrane retrieval, fluorescently tagged MAb 3C9 was colocalized with known endocytic markers with the use of fluorescence microscopy and gold-labeled MAb 3C9 was localized with electron microscopy. The use of the fluorescent probe 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene p-toluenesulfonate (TMA-DPH) as an endocytic pathway marker (15) enabled us to demonstrate that the retrieval of lamellar body membrane is different from bulk membrane retrieval. Our findings support the hypothesis that secretion and clearance of intra-alveolar surfactant may be connected through processes involving the lamellar body membrane.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Production of MAb 3C9. MAb 3C9 was raised by immunizing BALB/c mice with lamellar body membrane fractions isolated from rat lungs. Mouse sera and hybridoma supernatants were screened by indirect immunofluorescence of isolated type II cells. After 24 h in culture, MAb 3C9 labeled ringlike structures that were shown to be the limiting membrane of lamellar bodies in permeabilized, freshly isolated type II cells (32).

Labeling of MAb 3C9 and other markers. MAb 3C9, mouse IgG, and human or rat iron-saturated transferrin (Sigma, St. Louis, MO) were covalently labeled with fluorescein-EX or rhodamine with the use of the FluoReporter or Texas Red-X protein labeling kits (Molecular Probes, Eugene, OR). Fluorescein-conjugated, cationized ferritin from horse spleen was also purchased from Molecular Probes. Fluorescein-conjugated Chrom-Pure rat transferrin was from Jackson ImmunoResearch Laboratories (West Grove, PA).

MAb 3C9 and mouse IgG were conjugated with 10-nm colloidal gold (BBInternational, Cardiff, UK) by the addition of the minimum amount of MAb 3C9 or mouse IgG required to stabilize the gold in suspension. The antibody-gold conjugate was purified by centrifugation.

Lipids and liposome preparation. NBD-PC {1-palmitoyl-2-[12-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]dodecanoyl]phos- phatidylcholine}, dipalmitoylphosphatidylcholine (DPPC), egg phosphatidylcholine (PC), phosphatidylglycerol (PG), and cholesterol were obtained from Avanti Polar Lipids (Birmingham, Al). NBD-PC is a fluorescent analog of PC (excitation maximum, 463 nm and emission maximum, 536 nm) previously used to follow lipid uptake in the lung and in cultured type II cells (5, 23). The relative molar ratios of (NBD-PC)-DPPC-egg PC-PG-cholesterol used in the liposomes were 15:35:25:10:15. These ratios were chosen to reflect the lipid composition of the lung surfactant, with NBD-PC lipid replacing the fraction of DPPC in the mixture that maximizes liposome fluorescence (23). Uniform unilamellar liposomes with a diameter of 100-200 nm were prepared by extrusion through polycarbonate membranes (14).

In vivo experiments. Rhodamine-labeled MAb 3C9 or labeled mouse IgG (20 µg in 200 µl of 0.9% sodium chloride) was intratracheally instilled in an anesthetized rat. After 1 h on the respirator, the rat was killed, and the lung was perfused and fixed in 4% paraformaldehyde. A portion of the tissue was frozen and cryosectioned (8 µm), and another portion was embedded in Polybed 812 resin (Polysciences, Warrington, PA) for thinner sectioning. Some of the cryosections were also immunostained with a polyclonal antibody (NPROSP-C) against the amino terminus of the precursor peptide of the rat surfactant protein (SP) C, which has previously been shown to be specific to type II cells of the lung and is found in lamellar bodies (3). Antibodies to the precursor peptide were used because it has been difficult to raise antibodies to the highly hydrophobic mature SP-C (3).

Cell preparation. Type II cells were isolated from adult male Sprague-Dawley rat lungs according to the procedure of Dobbs et al. (8). Briefly, the lungs were perfused via the pulmonary artery with solution II (0.9% saline and 0.1% glucose with 10 mM HEPES, 5 mM KCl, 2.5 mM sodium phosphate buffer, 1.7 mM CaCl2, 1.3 mM MgSO4, 35 mg of penicillin, and 50 mg of streptomycin in 500 ml). Lungs were then lavaged eight times through a tracheal cannula with solution I (0.9% saline and 0.1% glucose with 10 mM HEPES, 5 mM KCl, 2.5 mM sodium phosphate buffer, 35 mg of EGTA, 35 mg of penicillin, and 50 mg of streptomycin in 500 ml) and two more times with warm solution II. For elastase digestion, 10 ml of solution II containing 3 U/ml of elastase were instilled into the trachea. The elastase instillation was repeated twice more at 10-min intervals. The lung was minced with a McIlwain tissue chopper in the presence of a small volume of 100% FBS (ICN/Flow Laboratories, ICN Biochemicals, Costa Mesa, CA) and then poured into 10 ml of solution II containing 4 mg of DNase (Sigma). Cells were separated by filtration through a sequence of nylon meshes (160, 37, and 10 µm). The remaining macrophages were removed by plating the cell suspension onto a rat IgG (Sigma)-covered petri dish for 60 min. The unattached type II cells were harvested and centrifuged at 1,000 rpm for 10 min, then resuspended in MEM containing 10% FCS. The cells were plated onto glass coverslips (Fisher Scientific, Pittsburgh, PA) or glass-bottomed dishes (MatTek, Ashland, MA) at a density of 1-2 × 106/35-mm culture dish. Cells were cultured overnight in a humidified 37°C incubator supplemented with 5% CO2 in air. Purity of the type II cell preparation was >92% as determined by modified Papanicolau stain.

Cell treatment for colocalization experiments. Type II cells plated onto glass coverslips were cultured for 18-24 h. Nonadherent cells were removed with several washes of MEM at room temperature. The plated cells were incubated with MEM containing 4 µg/ml of fluorescein-labeled MAb 3C9 and 4 µg/ml of transferrin from either Sigma or Jackson ImmunoResearch Laboratories. Internalization was stimulated with 0.1 mM ATP (Sigma), a known surfactant secretagogue. After various times of incubation, the internalization was stopped by washing the cells with ice-cold 0.05 M PBS followed by two quick washes with cold 5% acetic acid to remove surface-bound ligands. Cells were fixed with 4% paraformaldehyde for 10-15 min at room temperature.

For colocalization of MAb 3C9 and cationized ferritin or NBD-PC liposomes, cultured cells were depleted of serum for 1 h by exchanging the 10% FCS containing MEM for Hanks' balanced salt solution (HBSS). Cells were incubated in HBSS containing 4 µg/ml of rhodamine-labeled MAb 3C9, 0.2 mg/ml of fluorescein-labeled cationized ferritin, or 0.14 mg/ml of 15 mol/100 ml NBD-PC labeled liposomes and 0.1 mM ATP. Surface-bound ferritin was removed with a brief additional wash with cold 1 M sodium chloride.

Endocytosis of MAb 3C9 or TMA-DPH. Type II cells were plated onto 30-mm glass-bottomed dishes (MatTek) for 24 h. Each 30-mm dish was covered with a 100-mm dish through which a gas mixture of 5% CO2 and 95% O2 flowed. This simple environmental chamber was maintained at 37°C with a heat curtain (Sage Instruments, White Plains, NY) and imaged with an inverted microscope. The cells were incubated with 2 × 10-6 M TMA-DPH (Molecular Probes) or 4 µg of rhodamine-labeled MAb 3C9. After ATP and the marker to the cells were added, fluorescent images were acquired at 15-s intervals for TMA-DPH and at 2- to 15-min intervals after MAb 3C9 internalization.

Sequential labeling. Cells were allowed to internalize the antibodies labeled with two different fluorophors in sequence. Type II cells in culture, stimulated with ATP, were first incubated with rhodamine-labeled MAb 3C9 for 30 min followed by a 30-min incubation with fluorescein-labeled MAb 3C9. This resulted in a visible fluorescent signal from internalized, labeled MAb 3C9, but the intensity was low. To increase the amount of internalized MAb 3C9 and to keep the incubation time at a reasonable level, we also used a second method in which type II cells were incubated with rhodamine-labeled MAb 3C9 for 2 h without ATP stimulation, followed by incubation for 30 min with fluorescein-labeled MAb 3C9 in the presence of ATP. Similar results were obtained with both schedules.

Microscopy. All fluorescence microscopy was performed with a Nikon Diaphot inverted microscope equipped with a ×100 Fluor Phase 4 oil-immersion lens. Images were acquired with a cooled charge-coupled device camera (Princeton Instruments, Trenton, NJ) and Metamorph image processing software (Universal Imaging, West Chester, PA).

For electron microscopy, isolated type II cells cultured for 24 h were incubated with 3.5 µg/ml of colloidal gold-labeled MAb 3C9 for various times. The uptake was stopped with several washes with ice-cold HBSS, and the cells were fixed with gluteraldehyde. The cells were scraped from the dish, treated with OsO4, pelleted, and dehydrated with graded concentrations of cold acetone. The pellets were embedded in PolyBed (Polysciences), and ultrathin sections were cut. Electron micrographs were acquired with a JEOL 100CX microscope.

Data analysis. Results are reported as means ± SE unless otherwise stated. Results were analyzed by paired t-test with SigmaStat for Windows (Jandel, San Rafael, CA), and the level of significance was taken as P < 0.05.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Uptake of fluorescently labeled MAb 3C9. Previous work (32) showed that MAb 3C9 specifically labeled the membranes of lamellar bodies and multivesicular bodies in fixed and permeabilized freshly isolated lung type II cells and in type II cells cultured for 24 h, but it did not label alveolar macrophages. The epitope recognized by MAb 3C9 was found to be located on the interior surface of lamellar body membrane. It was predicted that after secretion of the contents of lamellar bodies, the MAb 3C9 epitope would be exposed to the alveolar surface of the type II cell. If the lamellar body membrane was then retrieved, an antibody bound to the epitope might also be retrieved. To determine the specificity of MAb 3C9 and the ability of lung type II cells to internalize this antibody in vivo, we instilled rhodamine-labeled MAb 3C9 intratracheally in several rats. The distribution of labeled MAb 3C9 in a representative cryosection of lung 1 h after instillation is shown in Fig. 1, A-C. Virtually all of the MAb 3C9 was found in the alveoli, with no detectable label in the larger airways. To determine the cell type that had internalized the MAb 3C9, a selection of lung sections were also labeled with an antibody to NPROSP-C (Fig. 1B). The NPROSP-C antibody had previously been shown (3) to exclusively label alveolar type II cells. All the cells that had internalized MAb 3C9 were also labeled with NPROSP-C (Fig. 1, A and B). Conversely, all cells in the region that were labeled with NPROSP-C also internalized MAb 3C9. A thinner section of the same lung embedded in epoxy resin (Fig. 1, D and E) provided a higher magnification image of the structure of a single alveolus. Labeling was found in alveolar epithelial cells where MAb 3C9 appeared to be internalized into large perinuclear structures (Fig. 1D), with no apparent labeling of the cell surface. Weak, diffuse labeling of larger cells, probably macrophages, in the alveolar lumen was also occasionally seen (data not shown). MAb 3C9 is probably taken up by macrophages through a nonspecific Fc receptor pathway. To follow the retrieval of lamellar body membrane by living type II cells, freshly isolated cells were plated onto glass-bottomed dishes for 24 h, incubated with rhodamine-labeled MAb 3C9 in an environmental chamber at 37°C, and imaged with an inverted fluorescence microscope. Time-lapse sequences of fluorescent images of cell clusters were acquired before and after the addition of MAb 3C9 (Fig. 2). Before (Fig. 2A) and immediately after (Fig. 2B) MAb 3C9 was added, the fluorescent signal was low. Antibody binding to the cell surface was observed 2 min after the addition of MAb 3C9 (Fig. 2C, arrow). This antibody binding is presumed to be surface-expressed lamellar body membranes. After 5 min of incubation, ringlike structures were labeled (Fig. 2D, arrows), corresponding to lamellar bodies in the phase-contrast image of the same cells (Fig. 2F, arrows). Labeling of punctate dots can be observed after 5 min (Fig. 2D) but especially after 10 min (Fig. 2E, arrowheads) of incubation. During the first 10 min of the time-lapse sequence, the total fluorescence and the number of labeled lamellar bodies continued to increase (Fig. 2, B-E). After 10 min of incubation, there was no further increase in either the total fluorescent signal or the number of labeled lamellar bodies within the observed cell cluster (data not shown). For cells outside the region that was exposed to the fluorescent excitation light, the fluorescent signal continued to increase after 10 min (data not shown), suggesting that some cell damage or dye bleaching occurred during the exposure to light. To visualize and measure the binding and internalization of MAb 3C9 without the effect of light exposure, cells cultured on glass coverslips were incubated with MAb 3C9 for various times, then were fixed and mounted for observation. In cells cultured for 24 h, the binding and internalization of MAb 3C9 increased during 90 min of incubation, whereas internalization of nonspecific rhodamine-labeled mouse IgG remained at a low level (Fig. 3). Stimulation with the secretagogue ATP resulted in a significant increase of MAb 3C9 internalization (Fig. 3) after 20 min of incubation.


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Fig. 1.   In vivo instilled monoclonal antibody (MAb) 3C9 is internalized into type II cells. A: a representative lung cryosection after instillation of fluorescently labeled MAb 3C9. B: lung cryosection after subsequent immunostaining with NPROSP-C. C: phase-contrast image of the same area as in A and B. Instilled MAb 3C9 and NPROSP-C colocalize in type II cells (A and B, arrows). Higher magnification images of MAb 3C9 fluorescence (D) and phase contrast (E) were obtained from a resin-embedded section of the same lung. Punctate perinuclear staining is observed in 3 type II cells in D. Scale bars, 10 µm.



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Fig. 2.   Internalization of MAb 3C9 by type II cells in culture. Fluorescence images (A-E) were acquired with identical exposures of a cluster of cells incubated with labeled MAb 3C9 for various times. F: phase-contrast image of the same area as in A-E, showing lamellar bodies (arrows). The fluorescent signal was low before (A) and immediately after (B) MAb 3C9 was added. Two minutes after the addition of MAb 3C9, binding to the cell surface was visible (C, arrow). D: labeling of lamellar bodies (arrows) and small punctate spots were detectable after 5 min of incubation. E: the intensity and the number of labeled lamellar bodies and punctate dots (arrowheads) continued to increase for 10 min after the addition of MAb 3C9.



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Fig. 3.   Internalization of MAb 3C9 can be stimulated with the secretagogue ATP. Cells plated on glass coverslips were incubated with fluorescently labeled MAb 3C9 or labeled nonspecific mouse IgG. Internalization of MAb 3C9 was significantly higher during stimulation with 0.1 mM ATP () than in unstimulated cells (). Values are means ± SD; n = 5 experiments. Fluorescence from type II cells that were incubated with labeled nonspecific mouse IgG remained at a low level. Stimulation with ATP had no effect (open circle ) on IgG uptake over control cells ().

Colocalization with endocytic markers. Cationized ferritin was previously shown (16, 30) to be internalized into endosomes, multivesicular bodies, and lamellar bodies of type II cells. In this study, cultured type II cells were incubated with rhodamine-labeled MAb 3C9 (Fig. 4A) and fluorescein-labeled cationized ferritin (Fig. 4B) for 2 h. The majority of cells internalized fluorescently labeled cationized ferritin, but not all cells internalized MAb 3C9 (data not shown). In cells that internalized both, the labels colocalized in ringlike structures (Fig. 4, arrows) that were presumed to be lamellar bodies and in punctate structures presumed to be endosomes, multivesicular bodies, or lysosomes (Fig. 4, arrowheads).


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Fig. 4.   Colocalization of MAb 3C9 and cationized ferritin in punctate dots and lamellar bodies. Shown are fluorescence images of a representative cluster of cells that had been incubated for 2 h with rhodamine-labeled MAb 3C9 (A) and fluorescein-labeled cationized ferritin (B). C: phase-contrast image of the same area as in A and B. MAb 3C9 and ferritin both label lamellar bodies (A and B, arrows) and punctate dots (A and B, arrowheads) in some cells. B: cationized ferritin is internalized uniformly by almost all the type II cells and MAb 3C9 is internalized by a fraction of the cells. In the fluorescent overlay of A and B (D), ferritin and MAb 3C9 colocalize in punctate dots (arrowhead) and in lamellar bodies (arrow), as indicated by the yellow color. Scale bar, 10 µm.

Transferrin is also internalized via receptor-mediated endocytosis, but it is recycled back to the cell surface through the sorting endosome. Cultured type II cells were incubated with rhodamine-labeled transferrin and fluorescein-labeled MAb 3C9 for 1.5 h. As with ferritin, transferrin and MAb 3C9 were not usually internalized in the same cell. The amount of internalized transferrin remained at a low level for all cells whether human or rat transferrin was used. When internalized into the same cells, transferrin and MAb 3C9 were found colocalized in punctate structures, but transferrin was not found with MAb 3C9 in lamellar bodies (data not shown).

Kinetics of bulk membrane retrieval in type II cells. TMA-DPH is a lipophilic, membrane-impermeant, fluorescent cationic lipid that fluoresces weakly in aqueous environments but fluoresces strongly when it is integrated into cell membranes. TMA-DPH has been used to trace endocytosis in a number of cell types (15). In type II cells, TMA-DPH incorporated into the plasma membrane within 2 min (Fig. 5B), and numerous bright fluorescent dots appeared in the cytoplasm after 5 min of incubation (Fig. 5C). The number of dots increased, and ringlike structures, presumed to be lamellar body membranes, appeared by 6 min (Fig. 5D) and continued to increase at 8 (Fig. 5E) and 10 (Fig. 5F) min after incubation. No further changes occurred during the next hour (data not shown). Occasionally, very bright and large (~1 µm) punctate structures stained with TMA-DPH (Fig. 5, B, D, and F, arrows). These structures resembled secreted surfactant that has been previously observed (13) with other lipophilic dyes. In co-uptake experiments, only a limited number of cells internalized MAb 3C9 (Fig. 6A), but TMA-DPH (Fig. 6B) was internalized into all type II cells in culture. In cells that internalized both, TMA-DPH and MAb 3C9 appeared to be present in the same lamellar bodies, with the MAb 3C9 image surrounding that of TMA-DPH. The solid TMA-DPH staining suggests that the dye transferred from the membrane to the lamellar body contents after 1 h (Fig. 6C).


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Fig. 5.   The endocytic marker TMA-DPH is internalized into lamellar bodies by type II cells. A: phase-contrast image of a representative cluster of cells that had been incubated with the lipophilic, membrane-impermanent, fluorescent cationic lipid TMA-DPH. TMA-DPH is integrated into the cell membrane after 2 min (B). TMA-DPH labels bright punctate spots and ringlike structures, presumed to be lamellar body membrane, after 5 (C) and 6 (D) min of incubation, and the number of spots and rings increases in all the cells at 8 (E) and 10 (F) min. TMA-DPH appears to label the solid contents of lamellar bodies that are being exocytosed (B, D, and F, arrows).



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Fig. 6.   The endocytic marker TMA-DPH colocalizes with MAb 3C9 in lamellar bodies. After 1 h of incubation with cultured type II cells, rhodamine-labeled MAb 3C9 stains the limiting membrane (A) and TMA-DPH labels the contents of the lamellar bodies (B). C: an overlay of A and B shows that MAb 3C9 staining surrounds the TMA-DPH labeling in the same organelle. Lamellar bodies appear as dark round structures in the paired phase-contrast image (D). Scale bar, 10 µm.

Colocalization of MAb 3C9 with internalized lipids. Liposomes that mimic the lipid composition of lung surfactant and that are labeled with trace amounts of fluorescent NBD-PC are internalized into the lamellar bodies of type II cells both in vivo and in vitro (5, 23). To test whether internalized lipid and retrieved lamellar body membrane are trafficked to the same organelles, cultured type II cells were incubated with both labeled liposomes and labeled MAb 3C9 in the presence of ATP for 2 h. The lamellar bodies of some of the cells contained MAb 3C9 (Fig. 7A); most of the cells contained NBD-PC (Fig. 7B). For those cells that internalized both markers, MAb 3C9 labeled the limiting membrane of NBD-PC-containing lamellar bodies (Fig. 7D).


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Fig. 7.   MAb 3C9 and liposomes containing NBD-PC are both reinserted into lamellar bodies. Rhodamine-labeled MAb 3C9 (A) is internalized to the limiting membrane of the lamellar bodies, whereas NBD-PC-containing liposomes are internalized to the interior of the lamellar bodies (B). C: phase-contrast image of the same area as in A and B. D: an overlay of A and B shows colocalization of MAb 3C9 and NBD-PC in the same lamellar body (A, B, and D, arrows). Scale bar, 10 µm.

Internalization of gold-labeled MAb 3C9 into type II cells in culture. The subcellular distribution of internalized gold-conjugated MAb 3C9 was determined with electron microscopy in order to identify the intracellular pathway by which lamellar body membrane is retrieved from the cell surface. The incubations were performed in the presence of ATP and were stopped after 2 (Fig. 8A), 5 (Fig. 8B), 10 (Fig. 8, C and D), 30 (Fig. 8, E and F), or 60 (Fig. 8G) min. Electron micrographs showed that the gold-conjugated MAb 3C9 is bound to the cell surface by 2 min (Fig. 8A) and at subsequent times afterward (Fig. 8, B, C, and F). Colloidal gold particles were found in electron-lucent vesicles at 5 min (Fig. 8B) and thereafter (Fig. 8F). Colloidal gold particles were found occasionally in lamellar bodies (data not shown), multivesicular bodies (data not shown), and composite bodies (Fig. 8E) after 10 min of incubation, but they were found far more frequently after 30 (Fig. 8F) and 60 (Fig. 8G) min of incubation. MAb 3C9 was also found, although rarely, in small coated vesicles inside type II cells (Fig. 8D, inset). The number of particles found in each lamellar body increased with time (Fig. 8, F and G) and appeared to cluster into patches (Fig. 8G). By 60 min, the retrieved MAb 3C9 was found in lamellar bodies that appeared to be in the process of secretion (Fig. 8G). To quantify the internalization of MAb 3C9, electron micrographs of 100 type II cells were selected based on the presence of intact lamellar bodies. Of the 100 cells randomly chosen, 35 contained at least one gold particle. In 100 type II cells similarly selected but that had been incubated with nonspecific mouse IgG-gold conjugates, no intracellular gold particles were found. Macrophages internalized both the MAb 3C9 and the nonspecific mouse IgG equally, indicating that macrophage labeling represented phagocytosis and not specific binding.


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Fig. 8.   MAb 3C9 is internalized into several compartments in cultured cells. Cultured type II cells were incubated with ATP and colloidal gold particles decorated with MAb 3C9 for 2 (A), 5 (B), 10 (C and D), 30 (E and F), and 60 (G) min. The cells were then fixed and prepared for electron microscopy. At the earliest time point, colloidal gold was found only on the cell surface. Gold particles were occasionally observed in coated vesicles by 5 min of incubation (D, inset) and were frequently observed in multivesicular bodies (mvb) or composite bodies by 30 min (E). After 30 min of continuous incubation with MAb 3C9, particles were found in lamellar bodies (lb; F) and still found in intracellular vesicles and tubular vesicular structures (F, arrows). Gold particles were found clustered on the inner membrane of a lamellar body (G, circles) that appeared to be undergoing secretion. Scale bar, 5 µm.

Sequential labeling of type II cells with MAb 3C9. Synaptic vesicle membranes remained intact throughout the secretion/retrieval cycle and were reutilized in the formation of new vesicles (24). To determine whether the lamellar body membrane was retrieved as an intact unit and then refilled to form a new lamellar body or if it was retrieved as smaller subunits and redistributed among the existing lamellar bodies, type II cells were sequentially incubated, first with rhodamine-labeled MAb 3C9 and then with fluorescein-labeled MAb 3C9. If the retrieval was of intact units, then the rhodamine and fluorescein labels would be present in a small number of different lamellar bodies. If, on the other hand, the retrieved membrane was redistributed among preexisting lamellar bodies, then the two labels would be present in the same lamellar bodies and in a larger number of lamellar bodies. For those cells that internalized both fluorophors, the two labels colocalized in the same ringlike structures (Fig. 9, A and B, arrows), again presumed to be lamellar bodies.


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Fig. 9.   Retrieved surface-expressed lamellar body membrane is reinserted into existing lamellar bodies. Shown is a representative cluster of cells incubated with rhodamine-labeled MAb 3C9 (A) for 2 h followed by a incubation with fluorescein-labeled MAb 3C9 (B) for 30 min. In cells that internalized both antibodies, the antibodies were integrated into the same lamellar body (arrows). C: paired phase-contrast image. Scale bar, 10 µm.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Type II cells of the lung are responsible for the synthesis, packaging, and secretion of lung surfactant. These same cells are also responsible for the uptake of surfactant components from the alveolar space and their reutilization for resecretion. The exocytosis and endocytosis of surfactant is coupled in type II cells; agents that increase secretion also increase uptake. As in other cases of regulated secretion, surfactant secretion occurs by the fusion of the limiting membrane of the secretory organelle (the lamellar body membrane in type II cells) to the cell surface and release of its contents into the extracellular space (18, 27). We have postulated that the uptake of surfactant located in the alveoli is coupled to the retrieval of the secretory granule membrane from the cell surface. After fusion of the membranes, the lipid contents of the lamellar body are slowly released from the surface of the cell, from a patch that previously was the interior surface of the lamellar body membrane. As demonstrated in an isolated cell assay (13), the release takes 10-20 min, suggesting that the lipid content is bound to the surface and that the surfactant in the lumen of the alveoli may also bind to the same patch of cell surface. As the lamellar body surfactant is released from the surface by mass action, the alveolar surfactant also binds to the surface by mass action, leading to a net exchange of the two pools at the membrane surface. When the lamellar body membrane is retrieved from the surface, the attached alveolar surfactant would be internalized into the cell and could be recycled back to preexisting lamellar bodies or utilized in the creation of new lamellar bodies.

To test some of these hypotheses, we generated a panel of antibodies to the limiting membrane of lamellar bodies to follow the trafficking of this membrane. One of these antibodies, MAb 3C9, is highly specific to type II cells, recognizing a 180-kDa lamellar body membrane protein, with an epitope inside the lamellar body. Because the interior of the lamellar body membrane is on the exterior of the cell after exocytosis, we were able to follow lamellar body membrane retrieval with the use of MAb 3C9.

Intratracheally instilled MAb 3C9 is internalized primarily by type II cells in vivo as shown by its colocalization with the type II cell-specific marker NPROSP-C. The internalized MAb 3C9 is in large punctate perinuclear structures likely to be lamellar bodies. The endocytosis of MAb 3C9 by cultured cells is similar to its uptake by cells in intact lung, although a smaller fraction of the cultured type II cells internalized MAb 3C9 than type II cells in intact lung. Nearly 100% of the cells in vivo that labeled with NPROSP-C internalized MAb 3C9, whereas 70% of the type II cells cultured on plastic internalized MAb 3C9. This is not surprising because the phenotype of type II cells changes significantly after their removal from the lung. During subsequent primary culture, type II cells lose their ability to produce mRNA for surfactant proteins, lose their lamellar bodies, and change their shape from cuboidal to flat within 3-5 days when plated on plastic dishes (19). If MAb 3C9 uptake were linked to secretion, then we would predict a concomitant decrease in MAb 3C9 uptake with culture. A decrease in uptake does occur after 24 h in culture, but it unexpectedly occurs at the level of individual cells. The data suggest that a fraction of the cells lose the capacity to secrete surfactant and subsequently lose the ability to endocytose MAb 3C9.

The internalization of fluorescently labeled MAb 3C9 by type II cells in culture increased with the addition of the secretagogue ATP. The uptake was time dependent, and the uptake began to saturate at ~1 h. The increased internalization of MAb 3C9 with ATP suggests that, as in other cell types, the membrane retrieval process is coupled to secretion (1, 24). From the time course of appearance of gold-labeled MAb 3C9 in various cell compartments, the pathway for uptake appeared to be 1) surface binding, 2) endosomes, 3) vesicular structures, possibly trans-Golgi network (Fig. 8F), 4) multivesicular bodies, and, finally, 5) lamellar bodies. After times longer than 10 min, colloidal gold particles appeared to accumulate in lamellar bodies and cluster on their membranes. Other organelles such as cis-Golgi, medial-Golgi, mitochondria, and lysosomes did not label with MAb 3C9. The absence of significant surface labeling of MAb 3C9 after 1 h of uptake during the in vivo experiments (Fig. 1) corroborates the observation of rapid retrieval of lamellar body membrane in the cultured cells.

Surface-expressed lamellar body membrane could be retrieved in several ways. For instance, membrane could be internalized in small fragments by clathrin- or non-clathrin-mediated endocytosis or in larger segments by a cytoskeletal-based mechanism (11). Gold particles were found only rarely in small coated vesicles or tubular vesicular compartments. These data suggest that if a large fraction of the MAb 3C9 antigen is retrieved by clathrin-mediated endocytosis, it must be rapidly removed from the endosome compartment and recycled back to lamellar bodies. The punctate labeling that is observed at early times of fluorescent MAb 3C9 uptake (Fig. 2) is likely to be due to trafficking of the antibody through the tubular-vesicular and multivesicular body compartments.

The cell culture model was partially successful in investigating the retrieval pathway by comparing the uptake of other endocytic markers to the retrieval of lamellar body membrane. To identify potential retrieval pathways, we compared the internal localization of MAb 3C9 with several endocytic markers, including transferrin, TMA-DPH, cationized ferritin, and NBD-PC within type II cells. Cationic ferritin is known to be internalized nonspecifically into endosomes, multivesicular bodies, lysosomes, and lamellar bodies of type II cells (16, 30), so it was not surprising that MAb 3C9 colocalizes with ferritin in a number of compartments. Unlike MAb 3C9, TMA-DPH appears to be retrieved nonspecifically into all the cultured type II cells, presumably by bulk membrane uptake (15). The lack of complete overlap of TMA-DPH and MAb 3C9 signals suggests that MAb 3C9 uptake is more specific. NBD-PC liposomes were internalized into lamellar bodies by a larger fraction of cultured type II cells than MAb 3C9. This suggests that, at least in the cell culture model, NBD-PC is internalized by other pathways besides lamellar body membrane retrieval. Although MAb 3C9 and NBD-PC are both internalized into lamellar bodies, they are localized to different parts of the lamellar body; MAb 3C9 labels the limiting membrane of lamellar bodies, whereas the lipid dye labels the contents. These data suggest that the antibody is not degraded and remains bound to its antigen for at least 60 min of the retrieval pathway as in other cell types (1). Transferrin colocalized at a very low level with retrieved MAb 3C9 and only in punctate dots, probably representing early endosomes because transferrin is not found in the degradative or synthetic pathway (7, 17, 22).

In cultured type II cells that internalized MAb 3C9, the label appeared to be uniformly distributed to most of the lamellar bodies in the cell. The number of gold particles observed in single lamellar bodies increased with time of exposure. If the lamellar body membrane is retrieved as an intact unit as in synapses (24), then the amount of MAb 3C9 in each lamellar body should remain constant. In addition, MAb 3C9 labeled with two different dyes and added to the culture medium sequentially was found in the same lamellar bodies in the cells. These two sets of data suggest that, at least in type II cells in culture, the retrieved lamellar body membrane is retrieved in smaller units and that these units are reinserted into preexisting lamellar bodies rather than being used to create new lamellar bodies. As mentioned previously (19), primary cultures of type II cells have a reduced ability to form new lamellar bodies. Although MAb 3C9 was retrieved into preexisting lamellar bodies for the cells used in this study, the membrane retrieval pathway for type II cells in situ (in the lung) must still be determined.


    FOOTNOTES

Address for reprint requests and other correspondence: H. Shuman, 1 John Morgan Bldg., Institute for Environmental Medicine, Univ. of Penn. School of Medicine, Philadelphia, PA 19104-6068 (E-mail: shuman{at}mail.med.upenn.edu).

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. Section 1734 solely to indicate this fact.

Received 19 October 1999; accepted in final form 28 March 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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