Generation and characterization of monoclonal antibodies to alveolar type II cell lamellar body membrane

K. Zen1, K. Notarfrancesco1, V. Oorschot2, J. W. Slot2, A. B. Fisher1, and H. Shuman1

1 Institute for Environmental Medicine, University of Pennsylvania Medical Center, Philadelphia, Pennsylvania 19104-6068; and 2 Department of Cell Biology, Utrecht University Medical School, 3584 CX Utrecht, The Netherlands

    ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References

Monoclonal antibodies against the limiting membrane of alveolar type II cell lamellar bodies were obtained after immunization of mice with a membrane fraction prepared from lamellar bodies isolated from rat lungs. The specificity of the antibodies was investigated with Western blot analysis, indirect immunofluorescence, and electron-microscopic immunogold studies of freshly isolated or cultured alveolar type II cells, alveolar macrophages, and rat lung tissue. One of the monoclonal antibodies identified, MAb 3C9, recognized a 180-kDa lamellar body membrane (lbm180) protein. Immunogold labeling of rat lung tissue with MAb 3C9 demonstrated that lbm180 protein is primarily localized at the lamellar body limiting membrane and is not found in the lamellar body contents. Most multivesicular bodies of type II cells were also labeled, as were some small cytoplasmic vesicles. Golgi complex labeling and plasma membrane labeling were weak. The appearance of lbm180 protein by immunofluorescence in fetal rat lung cryosections correlated with the biogenesis of lamellar bodies. The lbm180 protein decreased with time in type II cells cultured on plastic. The lbm180 protein is an integral membrane protein of lamellar bodies and was also found in the pancreas and the pancreatic beta HC9 cell line but not in the rat brain, liver, kidney, stomach, or intestine. The present study provides evidence that the lbm180 protein is a lung lamellar body and/or multivesicular body membrane protein and that its antibody, MAb 3C9, will be a valuable reagent in further investigations of the biogenesis and trafficking of type II cell organelles.

surfactant; multivesicular bodies; pancreatic beta -cell

    INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References

LAMELLAR BODIES, unique secretory granules of alveolar type II cells, play a central role in the synthesis, secretion, and reutilization of lung surfactant (13, 18, 19, 35). These organelles consist of densely packed lamellae surrounded by a limiting membrane (32). Because of the importance of the surface-active agent in lung function, the composition, structure, and function of the lamellar body content have been investigated extensively (35) and are composed primarily of phospholipid, with ~10% (by weight) of surfactant-associated and possibly other proteins. On the other hand, the limiting membrane of lamellar bodies has been poorly characterized. Without a lamellar body membrane-specific marker, processes such as the biogenesis and trafficking of lamellar bodies or the fate of the lamellar body membrane after surfactant release are difficult to follow. Recently, several studies (14, 30, 31) based on antigenic determinants have demonstrated that lamellar bodies share membrane proteins with lysosomes or late endosomes of other cell types. However, no membrane protein specific for lamellar bodies has been found.

In the present study, a panel of monoclonal antibodies was generated against a lamellar body membrane fraction purified from rat lung to find a marker for the limiting membrane of lamellar bodies. After 1,864 clones were screened against freshly isolated and cultured alveolar type II cells by immunofluorescence, 30 positive clones were found and 26 representative cell lines were selected for further propagation. Evidence presented here demonstrates that one of these monoclonal antibodies, MAb 3C9, recognizes a 180-kDa integral lamellar body membrane (lbm180) protein that is not found in other cells of the rat lung and is particularly localized at lamellar body and multivesicular body (MVB) limiting membranes of alveolar type II cells.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Materials. All chemicals used were analytic grade and obtained from either Fisher Scientific (Pittsburgh, PA) or Sigma (St. Louis, MO). All radioactive isotopes were obtained from Amersham (Arlington Heights, IL). Indocarbocyanine (Cy3)-conjugated affinity-purified goat anti-mouse IgG, FITC-conjugated affinity-purified goat anti-rabbit IgG, and horseradish peroxidase-conjugated affinity-purified goat anti-mouse IgG were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA). The fetal rat lung tissue was generously provided by Dr. L. Gonzales (Children's Hospital of Pennsylvania, Philadelphia). The rat pancreatic beta HC9 cell line was provided by Dr. F. M. Matschinsky (Diabetes Research Center, University of Pennsylvania, Philadelphia). The polyclonal antibody against the NH2 terminus of the rat surfactant protein (SP) C precursor protein (N-pro-SP-C) was provided by Dr. M. F. Beers (Institute for Environmental Medicine, University of Pennsylvania).

Preparation of lamellar bodies and lamellar body membrane. Lamellar bodies were isolated from rat lungs that had been cleared of blood with upward flotation on a sucrose density gradient as previously described (8). The lamellar body fraction was collected between 0.35 and 0.50 M sucrose and pelleted in 0.20 M sucrose by centrifugation at 20,000 g for 20 min. To isolate lamellar body membranes, the freshly isolated lamellar body fraction was suspended in a hypotonic solution (10 mM Tris · HCl-50 mM sucrose, pH 7.2) and set on ice for 3 h or overnight at 4°C. The suspension was loaded on a cushion of 0.50 M sucrose and centrifuged for 1 h at 25,000 g with a swinging-bucket rotor. The lamellar body membrane fraction was recovered from the pellet, whereas the lamellar body content fraction was recovered at the interface between the buffer and 0.50 M sucrose.

Cell preparation and culture. Alveolar type II cells were isolated from rat lungs with elastase digestion (12). Freshly isolated type II cells (after IgG panning) and overnight cultured type II cells were both used to screen the monoclonal antibodies. For freshly isolated cells, type II cells (2.5 × 106/dish) were plated on 100-mm plastic tissue culture dishes containing modified MEM with 10% FCS and 2.5 mM MnCl2 for 1 h at 37°C (22). In the presence of Mn2+, cells attached more rapidly to culture dishes. For cells cultured overnight, freshly isolated type II cells were plated on dishes containing MEM with 10% FCS and incubated at 37°C with 5% CO2. After overnight culture, the plates contained >90% type II cells (10). Alveolar macrophages were isolated from lung lavage according to the method of Bates and Fisher (2). Alveolar macrophages were cultured in MEM containing 10% FCS for 3 h at 37°C before use. Pancreatic beta HC9 cells were harvested from the culture dishes with a rubber policeman after the cells reached half-confluence and were then cytospun onto glass slides.

Preparation of monoclonal antibodies. Hybridomas were generated by the University of Pennsylvania Cell Center Service Facility. In brief, two BALB/c mice were immunized with lamellar body membrane fractions via intraperitoneal and subcutaneous routes alternately. Serum titer was monitored starting with the third injection, and the mouse with the higher titer as determined by indirect immunofluorescence of isolated type II cells was chosen for fusion. The spleen was dissected and homogenized with a Wheaton homogenizer with an 80-mesh screen. Erythrocytes were lysed by incubation in 5 ml of lysis buffer (ice-cold 0.17 M NH4Cl, pH 7.5) on ice for 8 min, and splenocytes were mixed with 3 × 107 Sp2/0Ag14 myeloma cells. Fusion was carried out essentially according to the procedure of Lane et al. (20) with Kodak PEG 1450 as the fusion agent. Fusion products were resuspended in 300 ml of complete H-Y medium consisting of 58% high-glucose DMEM, 7% NCTC 135 medium, 20% fetal bovine serum, 4 mM L-glutamine, 0.15 mg/ml of oxalacetate, 50 µg/ml of pyruvate, 0.2 U/ml of insulin, 5% Origen hybridoma growth supplement (Igen), 100 U/ml of penicillin, 0.1 mg/ml of streptomycin, 0.1 mM hypoxanthine, and 6 mM azaserine. The suspensions were distributed into twenty 96-well culture plates, incubated at 37°C in an atmosphere of 8% CO2 in air, evaluated for the presence of hybridomas between days 3 and 5, and fed with 100 µl of medium without azaserine and 2% Origen on day 6, and the supernatants were harvested for testing starting on day 10. Positive hybridoma wells were subcloned by limiting dilution, the number of clones in each well was determined by microscopic examination, and the subclones were rescreened.

Screening of antibodies. Indirect immunofluorescence of freshly isolated or overnight cultured rat type II cells was used for monoclonal antibody screening. Cells cultured on 100-mm tissue culture plates were fixed and permeabilized by treatment with cold methanol-acetone (1:1 vol/vol) for 2 min and were dried in air. A Berol China marker pen was used to draw a grid to divide the plates into 96 wells. About 20 µl of undiluted hybridoma culture supernatant from each clone were added to a well and incubated for 2 h at room temperature. The plates were washed three times with 0.5 M PBS buffer and then incubated with Cy3-conjugated goat anti-mouse IgG (1:200 dilution) in 0.5 M PBS buffer with 5% BSA and 10% normal goat serum. After 1 h of incubation, the plates were washed extensively with 0.5 M PBS buffer and scored by three independent examiners using fluorescence microscopy.

Purification and radioiodination of antibody. IgG was precipitated from the hybridoma supernatant with (NH4)2SO4 and iodinated with IODO-GEN (Pierce, Rockford, IL) (9). The iodinated IgG was dialyzed extensively against Tris buffer. The specific activity was 3,000 ± 420 counts · min-1 · ng protein-1, and >97% of the radioactive protein was precipitable with trichloroacetic acid. The iodinated IgG was kept at 4°C and used within 2 mo.

Electrophoresis and immunoblotting. Protein samples were separated with SDS-PAGE under reducing conditions. Samples were solubilized in sample buffer [125 mM Tris · HCl-0.32 M sucrose-2% (wt/vol) SDS-65 mM dithiothreitol-0.001% bromphenol blue, pH 6.8] at room temperature. The separated proteins were transferred electrophoretically onto nitrocellulose membranes (0.32-µm pore size; BA83, Schleicher & Schuell) overnight at 20 mA in transfer buffer [25 mM Tris base-192 mM glycine-20% (vol/vol) methanol, pH 8.0]. Protein binding sites were blocked in Tris-buffered saline (25 mM Tris base-192 mM glycine, pH 7.4) containing 1% gelatin for 1 h at room temperature, and the membranes were then incubated with shaking for 1 h at room temperature or overnight at 4°C in hybridoma culture supernatants diluted 1:10 in Tris-buffered saline with 0.5% Tween 20. The antigen-antibody complexes were visualized with horseradish peroxidase-conjugated goat anti-mouse IgG and enhanced chemiluminescence (ECL System, Amersham).

Immunofluorescence. The distribution of antibody binding sites was investigated with both isolated cells and rat lung tissues. For cell studies, alveolar type II cells with a low concentration of fibroblasts (freshly isolated or cultured overnight) and alveolar macrophages were fixed and permeabilized with cold methanol-acetone for 1-2 min and then rinsed with buffer A (0.5 M PBS) for 5 min at room temperature. Cells were incubated with undiluted hybridoma culture supernatants for 2 h at room temperature. After three washes with buffer A, cells were incubated with Cy3-conjugated goat anti-mouse IgG diluted 1:200 in buffer A containing 5% BSA and 10% normal goat serum (buffer B) for 1 h. For tissue studies, 5- to 8-µm cryosections of rat lung, stomach, and intestine were washed with buffer A and incubated in 0.1% NaBH4 for 5 min to reduce tissue autofluorescence. The sections were permeabilized with 0.3% Triton X-100 in buffer B for 30 min at room temperature and then incubated with undiluted hybridoma supernatant for 2 h at room temperature. After a 5-min wash with buffer B, sections were incubated with Cy3-conjugated secondary antibody diluted 1:200 in buffer B for 1 h at room temperature. After three washes, the sections were mounted in Mowiol and examined by fluorescence microscopy.

Double-immunofluorescence label. For double-immunofluorescence experiments, rat lung tissue sections were incubated with undiluted MAb 3C9 hybridoma supernatant and a polyclonal antibody (anti-N-pro-SP-C) against rat N-pro-SP-C (1:100 dilution) in buffer B for 2 h at room temperature. After a 5-min wash with buffer B, the sections were incubated with Cy3-conjugated goat anti-mouse IgG (1:200 dilution) and FITC-conjugated goat anti-rabbit IgG (1:100 dilution) in buffer B for 1 h at room temperature. After three washes, the sections were examined with fluorescence microscopy.

Microscopy. Samples were observed with an inverted Nikon fluorescence microscope equipped with a ×60 long-working-distance objective (Nikon) and a ×60 or ×100 oil-immersion objective (Nikon). The images of paired phase-contrast and fluorescence or of double-label fluorescence images were obtained with appropriate filter sets (Chroma, Burlington, VT). The average fluorescence intensity of individual cells in a tissue section was measured with MetaMorph image-analysis software (Universal Imaging, West Chester, PA).

Immunogold labeling with MAb 3C9. Rat lung tissue was obtained from animals that were perfusion fixed for 5-10 min, and the tissue was further immersed for 1-2 h in the fixative. Two fixatives were used: 1) 4% paraformaldehyde and 2) a mixture of 0.2% glutaraldehyde and 2% paraformaldehyde (final concentration), both in 0.1 M sodium phosphate buffer, pH 7.4. After fixation, the tissue was stored in 1% paraformaldehyde for an indefinite period. Before being cryosectioned, small tissue pieces were infused with 10% gelatin in PBS for 15 min at 37°C. Then, small blocks (>1 mm3) were cut at 4°C and immersed overnight in 2.3 M sucrose at the same temperature with gentle agitation. Ultrathin cryosections were prepared on a Leica (Vienna, Austria) Ultracut S/FCS with diamond knives (Drukker International, Cuijk, The Netherlands) as recently described (21). The sections were thawed and transferred to grids on drops of a 1:1 mixture of 2% methyl cellulose and 2.3 M sucrose or of 2.3 M sucrose alone. For lbm180 protein immunolabeling, the sections were incubated with MAb 3C9 (1:20) followed by rabbit anti-mouse IgG (Dako, Copenhagen, Denmark) and protein A-gold [10 nm (29)]. The sections were imaged with JEOL (Tokyo, Japan) electron microscope model 1200 EX or 1010.

Separation of integral membrane from bound proteins. Two methods were used to separate the proteins loosely bound to lamellar body membranes from the lamellar body integral membrane proteins. First, isolated lamellar body membrane fractions (1.0 mg/ml) were prepared in 150 µl of 10 mM Tris · HCl, pH 7.4, 150 mM NaCl, and 1.0% Triton X-114 at 0°C (6). The sample was then overlaid on a cushion of 6% (wt/vol) sucrose in Tris buffer (10 mM Tris · HCl, pH 7.4, 150 mM NaCl, and 0.06% Triton X-114). After incubation for 3 min at 30°C, the sample was centrifuged (300 g) for 5 min at room temperature. The hydrophilic proteins were recovered in the upper aqueous phase, whereas the amphiphilic integral membrane proteins were recovered in the lower detergent phase. Second, isolated lamellar body membranes were washed with a high pH solution (Na2CO3, pH 10-11). The membrane fraction and wash solution (supernatant) were both collected after centrifugation and immunoblotted with MAb 3C9.

Binding of MAb 3C9 to intact or disrupted lamellar bodies. A suspension of freshly isolated lamellar bodies (0.5 µg/µl) was divided into three aliquots. For binding to the intact lamellar body fraction, the first 50-µl aliquot of lamellar bodies was incubated with iodinated MAb 3C9 in isosmotic buffer (100 mM PBS-200 mM sucrose). For binding to the disrupted lamellar body fraction, the second 50-µl aliquot of isolated lamellar bodies was first suspended in hypotonic solution (100 mM PBS-50 mM sucrose) for 30 min (4°C) and then pelleted by centrifugation. The disrupted lamellar bodies were resuspended in isosmotic buffer (at the same concentration as for intact lamellar body fraction) and incubated with iodinated MAb 3C9 for 30 min at 4°C. The two suspensions were centrifuged at 14,000 g for 20 min. The pellets were washed two times with cold PBS buffer, and the radioactivity was measured with a gamma counter (WALLAC Wizard 1470-001, Turku, Finland). Counts in the two samples were normalized to the protein mass that was determined from the third aliquot of isolated lamellar bodies.

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

Lamellar body localization of antigen identified by MAb 3C9. With the use of a two-step method, 845 µg of a limiting membrane fraction from the lamellar bodies of 45 rats were purified and used for antibody production as described in METHODS. Initial screening of supernatants from hybridoma cultures revealed that out of 1,856 wells from 3 fusions, 30 contained antibodies that strongly labeled the membranes of the lamellar bodies of type II cells that had been cultured for 24 h on plastic. One of those monoclonal antibodies, MAb 3C9, labeled freshly isolated (Fig. 1, A and B) and 24-h cultured lung type II cells (Fig. 1, C and D) but did not label alveolar macrophages (Fig. 1, E and F) or lung fibroblasts (data not shown). In freshly isolated type II cells, MAb 3C9 labeled bright punctate structures (Fig. 1B) that appear to colocalize with phase-dense organelles in paired phase-contrast images (Fig. 1A). In type II cells cultured overnight, MAb 3C9 labeled bright ringlike structures (Fig. 1D, arrows) that appear to surround lucent vacuoles in paired phase-contrast images (Fig. 1C, arrows).


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Fig. 1.   Immunofluorescence of monoclonal antibody MAb 3C9 in freshly isolated alveolar type II cells (A and B), type II cells cultured overnight (C and D), and alveolar macrophages (E and F). A, C, and E are paired phase-contrast images of B, D, and F, respectively. In fresh cells, MAb 3C9 labels in a punctate pattern, whereas labeling in cultured cells has a ringlike pattern (C and D, arrows). Alveolar macrophages are not labeled by MAb 3C9. Bars, 10 µm.

To determine the location of MAb 3C9 staining in intact rat lung tissue, cryosections of adult rat lung were double labeled with MAb 3C9 and anti-N-pro-SP-C antibody (5). SP-C is produced exclusively in alveolar type II cells of the rat lung and processed in Golgi complex, MVBs and lamellar bodies (3, 30, 34). In these double-labeling experiments, N-pro-SP-C was used as a marker to independently identify alveolar type II cells in lung tissue. In tissue labeled with MAb 3C9 alone, MAb 3C9 specifically labeled cuboidal cells with a punctate pattern (Fig. 2B). Immunofluorescence images of tissue labeled with MAb 3C9 (Fig. 2D) and anti-N-pro-SP-C antibody (Fig. 2C) showed colocalization to alveolar type II cells. Because of the recently demonstrated presence of surfactant-like proteins in the rat gastrointestinal tract (27), the stomach and intestine were evaluated with immunofluorescence staining of cryosections but did not show specific labeling.


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Fig. 2.   Immunofluorescence labeling of rat lung cryosections with MAb 3C9 alone (A, phase-contrast image, and B, fluorescent image) and with both polyclonal antibody against NH2 terminus of rat surfactant protein C precursor protein (N-pro-SP-C; C) and MAb 3C9 (D). In rat lung labeled with MAb 3C9 alone, MAb 3C9 labels cuboidal cells with a punctate pattern. In tissue double stained with MAb 3C9 and anti-N-pro-SP-C, the 2 antibodies colabel type II cells. Bars, 10 µm.

Ultrathin cryosections of rat lung were immunogold labeled with MAb 3C9 (Figs. 3 and 4) to determine the intracellular localization of lbm180 protein. Gold particles were only found in type II epithelial cells, with very low background elsewhere (data not shown). Also, no significant labeling was observed in control sections that were subjected to the same immunoincubations but where MAb 3C9 was replaced with a nonrelevant monoclonal antibody (data not shown). In the type II cells, the major part of the lbm180 protein labeling was associated with lamellar bodies (Fig. 3A).


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Fig. 3.   Cryosections of rat lung fixed in glutaraldehyde-paraformaldehyde showing type II alveolar cells immunolabeled with MAb 3C9 and examined by electron microscopy. Major part of gold labeling is associated with lamellar bodies (lb). A: section thawed on methyl cellulose-sucrose mixture. Lamellar body profiles have irregular shape (see also Fig. 4). In some of them (at right and bottom), in which part of multilamellar content is preserved, immunolabeling seems to be suppressed. B and C: overstretched sections due to thawing on sucrose. As a result, lamellar body profiles are more rounded and lamellar body contents are not preserved in multilamellar condition but seem to leave behind amorphous spots inside and outside lamellar bodies. Lamellar body membrane protein with a molecular mass of 180 kDa (lbm180 protein) labeling of lamellar body membrane is often much more intense than in A but remains rather heterogeneous. In B, tubular myelin (tm) is not labeled, but plasma membrane facing alveolar lumen (al) shows some gold particles. However, lbm180 protein was usually undetectable in apical plasma as shown in C where a type II cell (II) faces an alveolar space collapsed during fixation. n, Nucleus; m, mitochondrion; g, Golgi complex; I, type I cell. Bars, 200 nm.


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Fig. 4.   Same as Fig. 3, but cryosections were fixed with paraformaldehyde. Predominant lamellar body labeling is clear in A. Multivesicular bodies (MVBs; star ) in A and B are labeled in peripheral membrane. In C, small lbm180 protein-positive membrane structures (arrows) are seen near lamellar bodies and MVBs. In B and C, Golgi cisternae and surrounding vesicles are weakly stained. Bars, 200 nm.

The gold labeling on the lamellar body membranes was quite heterogeneous in sections of glutaraldehyde-fixed tissue that had been thawed on methyl cellulose-containing drops. This thawing method prevents surface tension damage to fragile cellular structures (21). In particular, lamellar bodies maintained their natural shape, often retaining well-preserved lamellar contents. To see whether the retained contents caused the heterogeneous labeling, sections that were thawed on sucrose drops were also studied. This preparation resulted in a greater loss of the multilamellar contents and oversized lamellar body profiles due to the high surface tension of the sucrose solution (21). The lbm180 protein labeling of the sections thawed on sucrose was higher in some areas, but, in general, it was as heterogeneous as the sections thawed on methyl cellulose-containing drops (Fig. 3B).

The apical plasma membrane was usually devoid of gold particles (Fig. 3C). Occasionally, however, patches of gold particles were found on the cell surface, possibly representing sites of recent exocytosis of lamellar bodies (Fig. 3B). Like the lamellar body contents, the extracellular surfactant material that was sometimes encountered (Fig. 3B) showed no significant labeling. Intracellular lbm180 protein labeling apart from the lamellar bodies was less conspicuous and more clearly illustrated in tissue fixed without glutaraldehyde (Fig. 4). Cryosections prepared from paraformaldehyde-fixed tissue when thawed on methyl cellulose-sucrose mixtures (Fig. 4A) produced more intense labeling that was accompanied by good structural preservation. Most of the MVBs were positive for lbm180 protein (Fig. 4, A and B). MVB labeling was primarily on the outer membrane, with an intensity similar to that of the lamellar body membranes. Small lbm180 protein-positive vesicular structures occurred in the cytoplasm, particularly near the lamellar bodies (Fig. 4C). Weak labeling was sometimes observed in the Golgi complex (Fig. 4B), probably representing de novo synthesized lbm180 protein. Other intracellular organelles including nuclei, mitochondria, and endoplasmic reticulum were devoid of gold particles.

Biochemical characterization of antigen. The location of MAb3C9 binding was also examined with cell and subcellular fractionation. Western blot analysis (Fig. 5)demonstrated that MAb 3C9 recognized a single protein band with an apparent molecular mass of 180 kDa in alveolar type II cells (lane 1), but it was not present in lung lysosomes (lane 2) or alveolar macrophages (lane 3).


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Fig. 5.   Western blot analysis with MAb 3C9 of rat alveolar type II cells (lane 1), rat lung lysosomal fraction (lane 2), and rat alveolar macrophages (lane 3). Samples were loaded as 10 µg protein/lane for lysosomes and 40 µg/lane for cell lysate. MAb 3C9 recognizes a band at 180 kDa only in alveolar type II cells but not in lung lysosomal fraction or alveolar macrophages. Nos. on left, molecular mass in kDa.

To test whether MAb 3C9 recognizes a lamellar body membrane protein, the isolated lamellar body membrane and lamellar body content fractions were analyzed by Western blot (Fig. 6). MAb 3C9 recognized a single band at 180 kDa in the lamellar body membrane fraction (lane 3) but did not recognize any bands in the lamellar body content fraction (lane 4).


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Fig. 6.   A Coomassie blue 250-stained gel (lanes 1 and 2) and matching MAb 3C9 Western blot (lanes 3 and 4) of lamellar body membrane (lanes 1 and 3) and content (lanes 2 and 4) fractions. The 180-kDa protein band recognized by MAb 3C9 is found in lamellar body limiting membrane fraction but not in lamellar body content fraction. Nos. on left, molecular mass in kDa.

To determine whether MAb 3C9 recognizes an integral membrane protein, the proteins of lamellar body membranes were further separated into integral membrane protein and membrane-associated protein with two different methods. In the first method, the membrane fraction was treated with Triton X-114 (6). The integral membrane proteins were recovered from the detergent phase and the membrane-associated proteins from the aqueous phase. In the second method, the membrane fraction was treated with a high pH solution (Na2CO3) and centrifuged, and the integral membrane proteins were collected in the pellet, whereas the membrane-associated proteins were recovered in the supernatant. In Western blot analysis (Fig. 7), MAb 3C9 recognized a 180-kDa band in the protein fractions recovered from the detergent phase of the Triton X-114 extract (lane 1) and from the pellet after the high pH solution wash (lane 3), but only low levels of antigen were detected in the aqueous phase (lane 2) or supernatant from the high pH solution wash (lane 4). On the basis of these results, the 180-kDa protein that was recognized by MAb 3C9 was named the lbm180 protein.


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Fig. 7.   Western blots with MAb 3C9 of a lamellar body membrane fraction incubated with Triton X-114 or treated with Na2CO3. MAb 3C9 strongly reacts with proteins recovered from detergent phase (lane 1) but only weakly reacts with proteins from aqueous phase (lane 2). MAb 3C9 strongly reacts with proteins recovered from pellet (lane 3) after centrifugation of a high pH solution wash but reacts weakly with soluble protein (lane 4). Nos. on left, molecular mass in kDa.

The topology of the MAb 3C9 labeling of lamellar body membrane was determined by measuring the binding of iodinated MAb 3C9 to intact lamellar bodies or lamellar bodies after osmotic disruption in hypotonic medium (Table 1). The binding of 125I-labeled MAb 3C9 to disrupted lamellar bodies was about fourfold higher than that to intact lamellar bodies, suggesting that the antigenic site of lbm180 protein is located on the luminal side of the lamellar body membrane.

                              
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Table 1.   Binding of MAb 3C9 to intact and disrupted LBs

The lbm180 protein appears late in gestation and its level increases during lung maturation. A previous study (33) indicated that lamellar bodies appear in the rat fetal lung at 19 days of gestation. To compare the appearance of the lbm180 protein with the appearance of lamellar bodies, fetal rat lung tissue was evaluated with MAb 3C9 by fluorescence microscopy at different stages of development. In a 17-day fetal rat lung, staining with MAb 3C9 was uniformly weak throughout the whole tissue (Fig. 8B). In a 21-day fetal rat lung, staining with MAb 3C9 was specific to isolated groups of cells. In these cells, labeling was strong and limited to large granules or ringlike structures (Fig. 8D, arrows). Labeling of a 9-day postnatal rat lung (Fig. 8F) and a 30-day postnatal rat adult lung (data not shown) with MAb 3C9 was similar to that in the 21-day lung. The data suggest that the lbm180 protein appears in the fetal rat lung between 17 and 21 days of gestation.


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Fig. 8.   Immunofluorescence of cryosections of rat lung tissues at 17 days of gestation (A and B), 21 days of gestation (C and D), and 9 days postnatal (E and F) labeled with MAb 3C9. MAb 3C9 weakly labels fetal 17-day tissue in a uniform pattern but labels cuboidal cells (arrows) with a punctate pattern in fetal 21-day tissue and 9-day postnatal tissue. A, C, and E are paired phase images of B, D, and F, respectively. Bar, 10 µm.

To quantify the appearance of MAb 3C9 labeling during lung development, the average fluorescence intensity of labeled cells was measured with image-analysis software (Fig. 9). The average fluorescence intensity per cell of labeled cells in a 21-day fetal rat lung was about fivefold higher than in the cells in a 17-day rat lung. The average fluorescence intensity per labeled cell was the same in a 9-day postnatal rat lung as in a 21-day fetal rat lung.


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Fig. 9.   Quantitative analysis of MAb 3C9 labeling in developing rat lung. Average fluorescence intensity/cell (in arbitary units) was measured in 20 labeled cells from 4 different cryosections with image-analysis software. Data are means ± SE. 17D and 21D, 17 and 21 days of gestation, respectively; P-9D and P-30D, 9 and 30 days postnatal, respectively.

Decrease of lbm180 protein in alveolar type II cells during cell culture. When type II cells adhere to plastic in primary culture, they rapidly spread and lose their ability to synthesize and secrete surfactant. To determine whether type II cells lose lbm180 protein during cell culture, type II cells were labeled with MAb 3C9 after various times in culture. After 1 day of culture, MAb 3C9 strongly labeled lamellar bodies (Fig. 10B, arrows). After 3 days of culture, the number of lamellar bodies decreased, and MAb 3C9 labeling appeared to surround enlarged vacuoles (Fig. 10D, arrows). The enlarged vacuoles are presumed to be the remains of lamellar bodies (17) after lipid extraction. After 5 days, MAb 3C9 labeling in type II cells had disappeared (Fig. 10F). The data suggest that type II cells lose lbm180 protein during culture on plastic.


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Fig. 10.   MAb 3C9 labeling decreases in alveolar type II cells with time in cell culture. Alveolar type II cells cultured on plastic dishes were labeled with MAb 3C9 after 1 (B), 3 (D), and 5 (F) days in culture. A, C, and E are paired phase-contrast images of B, D, and F, respectively. In 1-day cultured type II cells, MAb 3C9 strongly labels lamellar bodies (A and B, arrows). In 3-day cultured type II cells, MAb 3C9 weakly labels a few large dilated lamellar bodies (C and D, arrows). Labeling by MAb 3C9 was not detected in 5-day cultured type II cells. Bar, 10 µm.

Distribution of antigen in other secretory tissues. To determine whether lbm180 protein is present in other secretory tissues, immunoreactivity of MAb 3C9 was tested in rat brain, liver, kidney, and pancreas. The MAb 3C9 identified a protein band with an apparent molecular mass < 180 kDa in the pancreas (data not shown) but not in any of the other tissues tested. The rat pancreatic cell line beta HC9 (26) was also tested. Western blot analysis showed that MAb 3C9 recognized a single band at 180 kDa in beta HC9 cell lysate (Fig. 11A), and immunofluorescence demonstrated that MAb 3C9 labeled beta HC9 cells in a punctate pattern (Fig. 11C, arrows).


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Fig. 11.   Labeling of MAb 3C9 in rat pancreatic beta HC9 cells. In Western blot (A) of beta HC9 cell lysate (40 µg protein/lane), MAb 3C9 recognizes a single protein band around 180 kDa. Nos. on left, molecular mass in kDa. C: in immunofluorescence image of beta HC9 cells, MAb 3C9 labels cells in a punctate pattern (arrows). Punctate labeling may correspond to insulin secretory granules. B: paired phase-contrast image. Bar, 10 µm.

    DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References

In the present study, a library of monoclonal antibodies that react with components of the limiting membrane of lamellar bodies has been prepared. One of those antibodies, MAb 3C9, recognizes a novel lamellar body membrane protein, lbm180 protein.

The lbm180 protein is an integral membrane protein of lamellar bodies. The lbm180 protein was characterized with Western blot analysis, immunofluorescence, and electron-microscopic immunogold histochemistry. All three techniques showed that lbm180 protein is specific to type II cells in the lung and is primarily localized to the limiting membrane of lamellar bodies and MVBs in those cells. With Western blotting, lbm180 protein was found only in a lamellar body fraction isolated from the lung but not in a lung lysosomal fraction or in alveolar macrophages (Fig. 5). Double-label immunofluorescence with MAb 3C9 and antibodies to N-pro-SP-C confirmed that lbm180 protein is specific to type II cells in lung cryosections (Fig. 2). In short-term culture of type II cells on plastic, the lamellar bodies become larger (17). When type II cells are fixed and the lipid components are extracted, large lucent structures, assumed to be the remains of lamellar bodies, are apparent in phase-contrast images. Immunofluorescence images of cells labeled with MAb 3C9 show that the fluorescence is localized to the edges of these phase-lucent structures.

These results were confirmed at the ultrastructural level in which electron-microscopic immunogold labeling showed preferential localization to lamellar bodies and that almost all lamellar bodies were labeled with MAb 3C9 (Figs. 3 and 4). The gold particles associated with the antibody were predominantly localized at the lamellar body limiting membrane but not at the multilayer structures inside the lamellar bodies, suggesting that the lbm180 protein is a protein of the limiting membrane of lamellar bodies. This result was confirmed by Western blot analysis with separated fractions of lamellar body membrane and content (Fig. 6). The binding assay with 125I-MAb 3C9 against the intact and disrupted lamellar body fractions (Table 1) suggests that the antigenic site of the lbm180 protein is located at the luminal side of the lamellar body limiting membrane. The relatively large degree of binding of the antibody to the intact lamellar body fraction may be due to the partial disruption of lamellar body membranes during isolation. Because the binding sites of MAb 3C9 to the lbm180 protein appear to be located at the luminal side of the lamellar body membrane, this site will be exposed to the extracellular space after the limiting membrane of a lamellar body fuses with the plasma membrane during surfactant secretion. MAb 3C9 should, therefore, be useful for investigating the fate of the lamellar body limiting membranes after lamellar body secretion.

Localization of lbm180 protein in other organelles of alveolar type II cells. In addition to lamellar bodies, lbm180 protein was found in MVBs (Fig. 4), in small vesicles near the lamellar bodies (Fig. 4C), and, weakly, in the Golgi complex (Figs. 3A and 4C). These subcellular organelles may be involved in the synthesis of lbm180 protein and its transport to lamellar bodies in type II cells. Like other membrane proteins of secretory granules (1), lbm180 protein may be processed in the Golgi complex and then sorted to lamellar bodies via vesicular transport. The transport from the Golgi complex to lamellar bodies along the synthetic pathway may be through acidic MVBs as previously found for the transport and processing of SP-C (3, 30).

Gold particles associated with MAb 3C9 were occasionally found clustered at the cell surface, and these patches were sometimes proximal to extracellular tubular myelin (Fig. 3B). The lbm180 protein could appear at the cell surface via two routes. In the first route, the lbm180 protein could be transported to the cell surface after synthesis, as with some lysosomal proteins (16). The lbm180 protein would then be internalized during endocytosis and delivered to lamellar bodies along the endocytic pathway. However, this mechanism might not explain the clustering of the antigen at the apical surface (Fig. 3B). In the second route, the lbm180 protein could appear on the cell surface after lamellar body secretion. During surfactant secretion, the lamellar body limiting membrane fuses with the cell-surface membrane, and surfactant is released into the extracellular space (18, 19). If the patch of the cell surface that corresponds to the lamellar body limiting membrane is not immediately retrieved, then some lbm180 protein should be found at the cell surface.

Correlation between generation of lbm180 protein and biogenesis of lamellar bodies. The correlation between the appearance of the lbm180 protein and lamellar bodies during lung development provides further support for the specificity of the lbm180 protein to lamellar bodies. In the fetal rat lung, fully differentiated alveolar type II cells with lamellar bodies that are evident morphologically appear on day 19 of gestation (33). This developmental timetable is in agreement with the expression of SP-A and SP-B proteins (4) and the expression of mRNAs for SP-A, SP-B, and SP-C (23, 28). Labeling of fetal rat lung with MAb 3C9 appears between days 17 and 21 of gestation, suggesting that the lbm180 protein appears in type II cells with the same developmental timetable as lamellar bodies (Figs. 8 and 9).

The disappearance of lbm180 protein from type II cells during cell culture also shows the connection between the lbm180 protein and lamellar bodies. Type II cells cultured on plastic rapidly lose their morphological and biochemical phenotype (11, 23). They do not make new surfactant proteins (24), and the preexisting lamellar bodies initially increase in size and then disappear. Labeling of type II cells with MAb 3C9 also disappears during cell culture (Fig. 10), again suggesting a relationship between the lbm180 protein and lamellar body genesis.

Distribution of lbm180 protein in other secretory tissues. Secretory granules from different tissues share several common characteristics such as the pathways used for sorting and transporting proteins to the granule, the proteolytic processing of the contents of the granules, and the mechanisms used for exocytosis (25). This raises the possibility that secretory granules share membrane proteins in common that may be responsible for these processes. Common secretory granule membrane proteins have been found in other tissues (1, 7, 15). In the present study, we have detected lbm180 protein in the pancreas and in pancreatic beta HC9 cells, a stable insulin-secreting cell line. The MAb 3C9 labeled the beta HC9 cells with a punctate pattern, suggesting the presence of lbm180 protein in the secretory granules of beta HC9 cells and indicating that lamellar bodies of alveolar type II cells share this membrane protein with the secretory granules of pancreatic beta -cells. The function of the lbm180 protein in either of these cell types remains to be determined.

In summary, by generating monoclonal antibodies directly against purified lamellar body limiting membranes, we have isolated 26 monoclonal antibodies that recognize the limiting membrane of lamellar bodies in immunofluorescence. One of these antibodies, MAb 3C9, has been further characterized and recognizes a 180-kDa integral membrane protein of lamellar bodies, lbm180 protein. On the basis of its specific intracellular localization, lbm180 protein can be used as a marker for the identification of lamellar bodies and their limiting membranes.

    ACKNOWLEDGEMENTS

We thank Dr. V. Bedian and the Cell Center of University of Pennsylvania (Philadelphia) for producing the monoclonal antibodies, Dr. M. F. Beers (Institute for Environmental Medicine, University of Pennsylvania) for the polyclonal antibody against the NH2 terminus of rat surfactant protein C precursor protein, Dr. L. Gonzales (Children's Hospital of Pennsylvania, Philadelphia) for the fetal rat lung tissue, Dr. F. M. Matschinsky (Diabetes Center, University of Pennsylvania) for the rat beta HC9 cells, Dr. Q. P. Chen for iodination of the 3C9 antibody, C. Dodia and J. Q. Tao for technical support, and Dr. S. Bates (Institute for Environmental Medicine, University of Pennsylvania) for suggesting the manganese method of rapid type II cell attachment.

    FOOTNOTES

Address for reprint requests: H. Shuman, 1 John Morgan Bldg., Institute for Environmental Medicine, Univ. of Pennsylvania School of Medicine, Philadelphia, PA 19104-6068.

Received 8 September 1997; accepted in final form 3 April 1998.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

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