The Relationship between Lamellar Bodies and Lysosomes in Type IT Pneumocytes Kevin F. Gibson and Christopher C. Widnell Division of Pulmonary/Critical Care Medicine and Department of Neurobiology, Anatomy, and Cell Science, University of Pittsburgh, School of Medicine, Pittsburgh, Pennsylvania
We have studied the relationship between lysosomes and lamellar bodies in alveolar type II (ATII) pneumocytes using a monoclonal antibody (anti-Igp-120) directed against a 120-kD rat lysosomal membrane glycoprotein and a polyclonal antibody (anti-SP-A) directed against rat surfactant protein A. The anti-Igp-120 precipitated a protein molecular mass of 120 kD from Triton celllysates radiolabeled with (3sS]methionine, and the anti-SP-A precipitated surfactant apoprotein A from the medium when analyzed under similar conditions. When ATII cells were cultured on Engelbreth-Holm-Swarm tumor basement membrane, and studied by indirect immunofluorescence, some structures seem to react with both antibodies, and others with only one. ATII cells cultured on plastic showed a major population of large vesicles that were labeled intensely with both antibodies, and a second population of vesicles that were labeled weakly and only with anti-SP-A. Analytical cell fractionation of freshly isolated ATII cells confirmed that Igp-120 was only present in structures containing the lysosomal matrix enzyme N-acetyl-{3-glucosarninidase. In contrast, SP-A was identified in two populations of vesicles with high phospholipid-to-protein ratios: one lacked N-acetyl{3-glucosarninidase and Igp-120and contained lamellar bodies; the other contained both lysosomal markers and a heterogeneous population of organelles that included multivesicular bodies, lamellar bodies, and lysosomes. Western blots of trichloroacetic acid precipitates of cell fractions identified proteins within the lysosomal compartment that reacted with anti-SP-A, but whose molecular mass was less than 28 kD. The results indicate that, in ATII cells, surfactant is located in two functionally distinct structures, one of which is probably involved in surfactant secretion, and the other, surfactant degradation. The techniques developed in this study should allow the role of these structures in the secretion and recycling of surfactant to be determined.
The protein and phospholipid constituents of surfactant are synthesized and secreted by alveolar type II (ATII) pneumocytes (I). However, this cell does not function simply in secretion, since several lines of evidence (2-4) indicate that the constituents of surfactant are taken up by the ATII cells and then reutilized. The presence of a receptor for surfactant protein A (SP-A) on the surface of the cells (5, 6) suggests that receptor-mediated endocytosis (7, 8) may be responsible for the uptake or that SP-A may regulate the secretion of surfactant (9). The intracellular events that result in the secretion of surfactant taken up by the cells remain to be established. Evi(Received in original form May 4, 1990 and in final form January 16, 1991) Address correspondence to: Kevin F. Gibson, M.D., Division of Pulmonary/Critical Care Medicine, Department of Medicine, University of Pittsburgh, School of Medicine, Pittsburgh, PA 15261. Abbreviations: alveolar type II, ATII; Engelbreth-Holm-Swarm tumor, EHS; endoplasmic reticulum, ER; fetal calf serum, FCS; Hepes-buffered saline + 10% FCS, HBS/FCS; phospholipid-to-protein ratio, Pl/Pr ratio; sodium dodecyl sulfate polyacrylamide gel electrophoresis, SDS-PAGE; surfactant protein A, SP-A. Am. J. Respir. Cell Mol. BioI. Vol. 4. pp. 504-513, 1991
dence obtained in studies of the intact lung indicate that the phospholipids of surfactant may recycle without being degraded (2-4, 10-14), while studies on cells in culture suggest that the phospholipids may be degraded and then resynthesized from the degradation products (15). The fate of the apoprotein moiety of surfactant has not been determined. Surfactant is stored within the ATII cells in lamellar bodies, which contain a multilamellar form of surfactant within a membrane-bound structure. How this secretory organelle is formed is not well understood, but it is likely that the surfactant phospholipids and apoproteins are synthesized separately in the endoplasmic reticulum (ER) where they are packaged into transport vesicles. The apoproteins undergo post-translational modification in the ER and Golgi before fusing with phospholipid-containing vesicles to form the mature lamellar body. This organelle may represent the functional equivalent of a storage granule in cells that exhibit regulated secretion of protein (16). In addition, lamellar bodies exhibit properties that are associated with organelles of the endocytic pathway in that they have been shown to contain lysosomal enzymes (17-21) and to fuse with multivesicular bodies (11, 12).
Gibson and Widnell: Lamellar Bodies and Lysosomes in Type II Pneumocytes
During the past few years, considerable progress has been made in characterizing the organelles of the endocytic pathway (22). Endocytic markers are transported first to early endosomes, where ligand-receptor complexes frequently dissociate; they then move to late endosomes, structures that frequently exhibit a multivesicular morphology and that contain lysosomal constituents at a lower concentration than that observed in lysosomes; and they are finally transported to mature lysosomes. If surfactant constituents are recycled without being degraded, they must be diverted from the endocytic pathway to the secretory pathway before degradation can occur. As a first step in analyzing the relationship between the endocytic and secretory pathways in ATII cells, we have characterized the organelles that contain SP-A, and a lysosomal glycoprotein (lgp-120) that is concentrated in lysosomes and also found in late endosomes (22-25). We show here that ATII cells synthesize this glycoprotein in vitro, and that it localizes to a lysosomal compartment. By isopycnic density gradient analysis, we show that two populations of organelles have high phospholipid-to-protein ratios and contain SP-A. The lighter population contains lamellar bodies that lack lysosomal constituents, whereas the denser population consists of multivesicular bodies, lysosomes, and other organelles. The techniques developed in this work should allow the intracellular fate of surfactant to be analyzed during recycling.
Materials and Methods Animals and Reagents Specific pathogen-free male Sprague-Dawley rats (250 to 275 g) were obtained from Zivic-Miller Laboratories and stored in a laminar flow hood prior to use. Ketamine HCI (100 mglml) was obtained from Parke-Davis (Morris Plains, NJ). Percoll, aprotinin, EDTA, N-ethylmalimide, Triton X-114, and fetal calf serum (FCS) were purchased from Sigma Chemical Co. (St. Louis, MO). Electrophoresis reagents were purchased from Bio-Rad (Richmond, CA). Sheep anti-rabbit IgG and fluorescein goat anti-rabbit IgG were obtained from Jackson Immunoresearch (West Grove, PA). Dulbecco's modified Eagle's medium was obtained from GIBCO (Grand Island, NY) and (3sS]methionine from Amersham (Arlington Heights, IL). Engelbreth-HolmSwarm tumor (EHS) basement membrane (Matrigel) was purchased from Collaborative Research (Bedford, MA) and used according to the manufacturer's specifications. 1251 was purchased from New England Nuclear (Boston, MA). A polyclonal antibody directed against rat SP-Awas a generous gift of G. Singh and S. Katyal. A monoclonal antibody directed against a 120-kD glycoprotein (anti-Igp-120) was a generous gift of I. Mellman. Cell Isolation and Culture Type II cell isolation was performed essentially as described by Weller and Karnovsky (26). This procedure yielded 2 x 107 cells/rat, 95 % of which were viable by trypan blue dye exclusion, and approximately 95 % of which were type II cells (confirmed by alkaline phosphatase staining [27], morphology, and the absence of indirect immunofluorescence with mouse monoclonal anti-rat leukocyte common antigen [26]). The plating efficiency averaged ""35%. Cells used in
505
metabolic studies were plated on 35-mm petri dishes (coated 30 min earlier with 200 p.l of EHS basement membrane) in Dulbecco's modified Eagle's medium + 10% FCS + antibiotics at a density of 1 x lQ6 cells/mm-. Indirect immunofluorescent studies were conducted on cells cultured on Labtek slides precoated with 50 p.l of EHS basement membrane per chamber (Miles Scientific, Naperville, IL). Cells were stained for alkaline phosphatase activity using an alkaline phosphatase staining kit (Sigma) and were routinely cultured at 1 x 105 cells/mm-. Indirect Immunofluorescence Cells were cultured for 3 d with or without EHS basement membrane prior to staining. Cells were fixed in 3.7% formaldehyde in phosphate-buffered saline at room temperature for 15 min and permeabilized with acetone at -150 C for 2 min. The cells were incubated for 1 h sequentially with Hepesbuffered saline + 10% heat inactivated FCS (HBS/FCS), rabbit anti-rat surfactant protein anti-serum (anti-SP-A) (10 p.l/ml)(28), anti-Igp-120 (1:100 dilution), rat anti-mouse IgG preabsorbed with rabbit serum (50 p.glml) , rhodamine mouse anti-rat IgG (50 p.glml) , and fluorescein-conjugated goat anti-rabbit IgG (50 p.g/ml), with three washes of HBS/FCS between steps. No significant differences were noted if the order of incubation of the fluorescent conjugate antibodies were changed, and no fluorescence was detected at identical exposures if the primary antibodies were omitted. The cells w~re mounted and photographed with an Olympus BH-2 epifluorescence photomicroscope equipped with filters to visualize fluorescein and rhodamine fluorescence. Cell Fractionation Analytical cell fractionation was conducted according to the methods of Tulkens and associates (29). Freshly isolated type II pneumocytes (2 to 3 X 108 cells) were suspended in 0.5 ml of 0.25 M sucrose containing 5 mM Hepes + 0.1 mM EDTA at 4 0 C The cell suspension was then homogenized with 10 strQ~es using a Dounce homogenizer and a B-type pestle, centnfuged at 500 X g for 10 min at 4 0 C, and the supernatant removed. The pellet was rehomogenized in 0.5 ml of the same medium, and the procedure was repeated until a postnuclear supernatant of 2.5 ml was obtained. Low temperature, calcium chelators, and a gentle disruption procedure ensured that lysosomal disruption was minimized during the homogenization procedure. When lysosomal disruption occurred (as was the case in earlier experiments), the enzyme markers were found in both heavy fractions and in at the top of the gradient with other soluble proteins. After removing 0.5 ml to measure homogenate activities, 2.0 ml o~ the postnuclear supernatant (approximately 25 mg of protem) was loaded on a 0.8 to 2.0 M sucrose linear gradient and centrifuged at 100,000 x g for 4 h at 4 0 C in a VTi 50 rotor (Beckman). In experiments using Percoll density gradients, the postnuclear supernatant was brought to a concentr~tion of 10% Percoll to loading on a 25% Percoll gradient WIth a 2.0 M sucrose cushion. The gradients were centrifuged at 70,000 X g for 30 min at 4 0 C in a VTi 50 rotor. Fractions were collected at 4 0 C with a Model 640 fracti?n collector (ISCO) and assayed for N-acetyl-fj-glucosamimdase (30), 5'-nucleotidase (31), succinate dehydrogenase
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(30), NADPH-cytochrome c reductase (32), galactosyl transferase (33), phospholipid-to-protein (PlIPr) ratios (see below), and refractive index (refractometer; American Optical Corporation). Enzyme assays were always carried out under conditions where product formation was linear with respect to time and the amount of cell fraction added to the assay. Enzyme activity recovered in subcellular fractions ranged from 85 to 100 % when compared to the homogenate enzyme activity. Phospholipids were extracted according to the methods of Folch and colleagues (34), and the phospholipid concentration measured according to the methods of Chen and coworkers (35) using egg lecithin as a standard. Protein was determined by the method of Lowry and associates (36). Values reported represent the means of two separate determinations. PlIPr ratios are not reported for fractions obtained on Percoll gradients because of background interference by Percoll. Fractions (""20 % of the total collected) to be analyzed for the presence of Igp-120 and SP-A (200 p,l) were first suspended in a 1:1 dilution of a solution containing 1% Triton X-114 + 150 mM NaCI + 10 mM Tris + aprotinin (0.23 U/ml) + 0.1 mM phenylmethylsulfonyl fluoride + 0.1 mM EDTA at 4 0 C and then vacuum-blotted (Slot blot; Hoefer Scientific Instruments) onto nitrocellulose. (The additional antiproteases were added to ensure that no protein degradation occurred after the vesicles were disrupted with Triton.) After incubation with HBS/FCS for 30 min, the membranes were incubated sequentially with rabbit anti-SP-A (1 p,lIml) for 1 h (to analyze SP-A), or anti-lgp-120 (1:1,000 dilution) and rabbit anti-mouse (1 p,g/ml) for Igp-120 at room temperature. The membranes were rinsed with HBS + 0.005 % Tween" 80 after each antibody incubation. The membranes were then incubated with 12S1 sheep anti-rabbit IgG (5 X lOs cpm/ml), visualized with autoradiography, and quantitated with scanning densitometry (GS 300 scanning densitometer; Hoefer). Densitometry results were linear with respect to the amount of antigen blotted and were expressed as a percentage of the homogenate activity. Electron Microscopy Pooled fractions obtained by analytical cell fractionation of freshly isolated ATII cells were fixed in suspension with 2.5% glutaraldehyde in cacodylate buffer (0.1 M cacodylate + 5 mM MgCl z + 1 mM CaCl z + 2 % sucrose) at 4 0 C for 1 h. Specimens were centrifuged at 100,000 X g for 1 h using an SW50 rotor (Beckman) and washed several times with buffer. The pellets were postfixed in 1% osmium tetroxide in cacodylate buffer, dehydrated with graded alcohol, and embedded in epon. Thin sections were stained with lead citrate and uranyl acetate and photographed with a Jeol JEM 100CX transmission electron microscope. Immunoprecipitation and Electrophoresis Cells cultured on 35-mm dishes were incubated with medium containing (3sS]methionine (100 p,Ci/ml) + 3 % FCS for 3 h at 37 0 C in 5 % CO z• The cells were then washed several times with fresh medium for 1 h. The cells were harvested with dispase (Collaborative Research) according to the manufacturer's instructions. Immunoprecipitation of Igp-120 from Triton X-114 cell homogenates was
performed according to the methods of Lewis and colleagues (23). Immunoprecipitated proteins were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE) (37) using 7.5 % gels and visualized by fluorography (AMPLIFY; Amersham). Immunoprecipitation of SP-A was conducted in a similar fashion using Triton X-100 instead of Triton X-114 and analyzed by SDS-PAGE using 10 to 15% gradient gels. In some experiments, trichloroacetic acid precipitates of cell fractions were evaluated by SDS-PAGE on 7.5 to 15% gradient gels under reducing conditions according to the method of Laemmli (37). Bovine serum albumin (100 p,glml) was added as a carrier prior to acid precipitation. After electroblotting onto nitrocellulose, SP-A was labeled with rabbit anti-SP-A and 1zS1 sheep anti-rabbit IgG and visualized with autoradiography as described above. Other Methods Sheep anti-rabbit IgG was iodinated according to methods of Fraker and Speck (38) and was affinity-purified on a rabbit IgG-Sepharose column prior to use.
Results Type II Pneumocyte Isolation and Culture For these experiments, we selected an isolation procedure that employs both a density gradient centrifugation step and a panning technique in order to obtain a highly purified cell preparation (26). In our first experiments, we studied the behavior of cells isolated by this technique in culture. Cells cultured on EHS basement membrane were rounded in appearance (Figure la) and stained positive for alkaline phosphatase (Figure lc), an enzyme found in high concentration in the plasma membrane of differentiated ATII cells (27). The cells formed aggregates early in culture, but after 3 d, the apical surfaces were still directed toward the overlying medium. After 7 d in culture, the morphologic features of each cell were less distinct as the cell aggregates enlarged, forming cystlike structures as they embedded in basement membrane (not shown). This has been previously described and occurs with other epithelial cells cultured on an EHS basement membrane matrix for extended periods (39,40). The formation of these cystlike structures was not detected before 3 d in our cell preparations. When cultured on plastic, ATII cells rapidly spread and adopted the appearance of squamous cells (Figure lb), The intensity of alkaline phosphatase staining decreased progressively and was scarcely detected at 3 d (Figure 1d). Only an occasional rounded cell could be identified that stained positively for this enzyme. As they dedifferentiated, the nuclei and nucleoli of the cells became more prominent and the large cytoplasmic organelles that conferred a granular appearance to freshly isolated cells gradually disappeared. Synthesis of SP-A and Igp-120 In Vitro Cells cultured on EHS basement membrane were studied to determine if they synthesized surfactant apoproteins and lgp120. After the third day in culture, they were radiolabeled with (3sS]methionine, lysed with a detergent buffer, immunoprecipitated with anti-lgp-120 and anti-SP-A, and analyzed as described in MATERIALS AND METHODS. As Figure
Gibson and Widnell: Lamellar Bodies and Lysosomes in Type II Pneumocytes
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c
Figure 1. Type ITpneumocytes were cultured for 3 d on Engelbreth-Holm-Swarm tumor (EHS) basement membrane (a and b) or tissue culture grade plastic (c and d), fixed, and stained according to methods described in MATERIALS AND METHODS. (a and c) Nomarski differential interference contrast microscopy; (b and d) phase-contrast images after alkaline phosphatase staining. Cells cultured on EHS basement membrane were more rounded in appearance and stained positively for alkaline phosphatase. When cultured on plastic, the cells rapidly spread, adopting the appearance of squamous cells. The intensity of staining of alkaline phosphatase decreased progressively in these cells, and most cells showed no detectable activity (bar = 16 Itm).
2 demonstrates, both SP-A and lysosomal integral membrane glycoprotein 19p-120 are synthesized by type II pneumocytes in culture. Anti-SP-A precipitated a heterogeneous group of proteins ranging in molecular mass between 26 and 36 kD, representing varying degrees of post-translational glycosylation of the protein (28, 41). The most prominent bands were at 32 and 38 kD. The heavily sialated lysosomal glycoprotein migrated as a broad band with an average M, of 120 kD, when analyzed under reducing conditions (23). Indirect Immunofluorescence Studies of SP-A and Igp-120 SP-A in cells cultured on EHS basement showed a diffuse dense pattern of immunofluorescence similar in appearance . to cells stained in situ (Figure 3a) (41). Staining patterns typical of individual organelles were hard to detect in these cells because their cuboidal shape caused the images of organelles to become superimposed, limiting their resolution at the light-microscope level. The pattern of fluorescence of 19p120 (Figure 3b) appeared more punctate, with some areas that overlapped with SP-A fluorescence; however, there were structures that apparently stained with either anti-SP-A or anti-lgp-120 but not both antibodies. The pattern of staining raised the possibility that there might be two populations of SP-A-containing structures (presumed lamellar bodies), one of which contained 19p-120 while the other lacked the lysosomal glycoprotein.
A Figure 2. Synthesis of surfactant protein A (SP-A) and Igp-120. Type II pneumocytes cultured for 3 d on EHS basement membrane were radiolabeled with [35S]methionine. Immunoprecipitates of SP-A (A) and Igp-120 (B) from detergent cell lysates were analyzed according to the procedure described in MATERIALS AND METHODS.
B
In order to better resolve organelles at the lightmicroscope level, the localization of SP-A and 19p-120 was investigated in cells cultured on tissue culture grade plastic (Figures 3c and 3d). These cells were much more spread out. When double-stained with the two antibodies and studied by fluorescence microscopy, large vesicular structures in the perinuclear region stained with both antibodies. All structures that stained positively for 19p-120 (Figure 3d) also stained for SP-A (Figure 3c). In addition, there was a very fine, and somewhat hard to detect, punctate immunofluorescence in cells stained for SP-A (Figure 3c), which did not appear to localize to the plasma membrane (i.e., not in the plane of focus of the plasma membrane) and did not label with Igp-120. Analytical Fractionation of Type II Pneumocytes The intracellular location of SP-A and Igp-120 was also investigated by analytical cell fractionation. Freshly isolated cells were studied to avoid any possible redistribution of markers that might occur during culture. Cell homogenates were fractionated on continuous linear sucrose gradients, and fractions were analyzed for PI/Pr ratios, 5'-nucleotidase, N-acetyl-J3-glucosaminidase, succinate dehydrogenase, NADPH-cytochrome c reductase, galactosyl transferase, SP-A, and Igp-120. The distribution of phospholipid and protein is shown in Figure 4a. There were two populations of organelles that contained high PlIPr ratios; one peak appeared at a density of 1.05 (0.55 M sucrose), while the second peak appeared at a density of 1.10 (1.19 M sucrose). The high PlIPr ratios identify structures that contain surfactant phospholipids; the PlIPr ratio of the postnuclear supernatant averaged 4.3 ~mollmg.
-
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Figures 4b and 4d show the distribution of lysosomal markers. Approximately 84% of the N-acetyl-J3-glucosaminidase activity was recovered at densities ranging between 1.13 and 1.20 (1.03 to 1.58 M sucrose), with a peak activity occurring at a density of 1"\.11.16 (Figure 4a). The lysosomal glycoprotein Igp-120 was recovered in the same fractions that contained N-acetyl-J3-glucosaminidase (Figure 4b). The figures shown are representative graphs obtained from one of four separate experiments that yielded qualitatively simi-
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AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 4 1991
Figure 3. Double indirect immunofluorescent staining of SP-A and Igp-120 in type II pneumocytes cultured on EHS basement membrane (a and b) (bar = 8 /Lm) or tissue grade plastic (c and d) (bar = 16 /Lm). Cells cultured on EHS basement membrane showed a diffuse dense pattern of immunofluorescence when stained for SP-A. The pattern of fluorescence of Igp-120 appeared more punctate, with areas that appeared to overlap with SP-A fluorescence; however, there were some structures that apparently stained with either anti-SP-A or anti-lgp-120 but not both antibodies. In cells cultured on plastic, large vesicular structures in the perinuclear region stained with both antibodies (arrows). In addition, there was a very fine and hard to detect punctate immunofluorescence in cells stained for SP-A which was not in the plane of focus of the plasma membrane and did not label with Igp-120.
lar results. The findings are consistent with previous observations that Igp-120 is localized primarily to late endosomal (prelysosome) and lysosomal membranes (23, 24, 42). In addition, the lighter portion of the lysosomal fraction appeared to overlap with the second PI/Pr peak, consistent with the presence of surfactant phospholipids within these structures. However, the light PI/Pr peak lacked lysosomal markers. The amount of SP-A (Figure 4c) was also assayed in each fraction , and the distribution showed two distinct peaks that were associated with high PI/Pr ratios. One peak occurred at a density of 1.05 (0.55 M sucrose) in a region devoid of N-acetyl-{3-glucosaminidase and Igp-120 and represented a region of the gradient where lamellar bodies are reported to migrate (43). In addition, there was a second peak at a density of 1.11 to 1.20 that co-localized with Igp-120and N-acetyl-{3glucosaminidase, suggesting that a population of vesicles bearing lysosomal markers (lgp-120 and N-acetyl-{3-glucosaminidase) and an elevated PI/Pr ratio, also contained SP-A. The percentage of SP-A recovered in fractions containing lysosomal markers varied from N60 to 90% of the total SP-A recovered in each experiment. The distribution of 5'-nucleotidase (plasma membrane), NADPH-cytochrome c reductase (ER), succinate dehydrogenase (mitochondria), and galactosyl transferase (Golgi) was also assayed, and the results are shown in Figure 4. 5'-nucleotidase (Figure 4e) and galactosyl transferase (Figure 4g) were detected in fractions with densities of 1.10 to 1.15, a region that partially overlaps with the lysosomal markers. NADPH-cytochrome c reductase (Figure 4h) and succinate dehydrogenase (Figure 4f) activities appeared in denser fractions, with density ranging between 1.113 and 1.20. None of these markers coincided with the light fraction that contained SP-A and a high PI/Pr ratio. Fractions from the lamellar body peak (labeled I in Figure 4) and the light (II) and dense (II) lysosomal peaks were pooled and studied by electron microscopy. Most of the structures in pooled fraction I consisted of lamellar bodies (Figure Sa). Many were surrounded by a distinct membrane and apparently intact (Figure 5b). Others, however, appeared partly disrupted-perhaps during preparation for
electron microscopy, and there were also smooth vesicles in the fraction (Figure Sa). The constituents of pooled fraction II are shown at low magnification in Figure 6a. This fraction contained vesicles, multivesicular bodies, lamellar bodies, mitochondria, Golgi stacks, and lysosomes. The multivesicular bodies were packed with vesicles and, in some instances, clearly identifiable lamellae (Figure 6b). Some contained vesicles and lamellae in an electron-dense matrix characteristic of lysosomes (Figure 6c). Pooled fraction ill (Figure 7) contained mostly dense lysosomes and mitochondria. In order to determine if the SP-A associated with the dense fractions was undergoing degradation, trichloroacetic acid precipitates of the fractions were analyzed by Western blot. As is shown in Figure 8, most of the SP-A detected in fractions with a density of 1.088 to 1.100 appeared to be intact protein (Figure 8b, I). However, in the more dense fractions (1.115 to 1.185), there was a smear of low-molecularweight proteins that were labeled by the antibody, suggesting that SP-A is undergoing degradation in these fractions (Figure 8b, 2 and 3). The amount of degraded apoprotein comprised a significant percentage of the total protein detected on the immunoblot and suggests that the apoprotein is present in, and being degraded by, Iysosomes. Analytical cell fractionation was carried out using Percoll density gradients in order to better resolve structures in the high-density regions . As Figure 9 demonstrates, two populations of vesicles that contained N-acetyl-{3-glucosaminidase and Igp-120 were detected at densities of Nl.l0 and N1.35. In addition, SP-A was associated with three distinct peaks (Figure 9c); only the second and third peaks contained 19p120 and N-acetyl-{3-glucosaminidase (Figures 9b and 9c).
Discussion The purpose of this study was to examine the role of the lysosome in the recycling of surfactant by ATII cells. We studied the distribution of SP-A in ATII cells in culture by immunofluorescence; cells cultured on EHS basement membrane showed SP-A in a diffuse staining pattern with areas that did not co-localize with Igp-120. In contrast, most of the
Gibson and Widnell: Lamellar Bodies and Lysosomes in Type II Pneumocytes
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We have studied the relationship between lysosomes and lamellar bodies in alveolar type II (ATII) pneumocytes using a monoclonal antibody (anti-Igp-120) directed against a 120-kD rat lysosomal membrane glycoprotein and a polyclonal antibody (anti-SP-A) directed against rat surfactant protein A. The anti-Igp-120 precipitated a protein molecular mass of 120 kD from Triton celllysates radiolabeled with (3sS]methionine, and the anti-SP-A precipitated surfactant apoprotein A from the medium when analyzed under similar conditions. When ATII cells were cultured on Engelbreth-Holm-Swarm tumor basement membrane, and studied by indirect immunofluorescence, some structures seem to react with both antibodies, and others with only one. ATII cells cultured on plastic showed a major population of large vesicles that were labeled intensely with both antibodies, and a second population of vesicles that were labeled weakly and only with anti-SP-A. Analytical cell fractionation of freshly isolated ATII cells confirmed that Igp-120 was only present in structures containing the lysosomal matrix enzyme N-acetyl-{3-glucosarninidase. In contrast, SP-A was identified in two populations of vesicles with high phospholipid-to-protein ratios: one lacked N-acetyl{3-glucosarninidase and Igp-120and contained lamellar bodies; the other contained both lysosomal markers and a heterogeneous population of organelles that included multivesicular bodies, lamellar bodies, and lysosomes. Western blots of trichloroacetic acid precipitates of cell fractions identified proteins within the lysosomal compartment that reacted with anti-SP-A, but whose molecular mass was less than 28 kD. The results indicate that, in ATII cells, surfactant is located in two functionally distinct structures, one of which is probably involved in surfactant secretion, and the other, surfactant degradation. The techniques developed in this study should allow the role of these structures in the secretion and recycling of surfactant to be determined.
The protein and phospholipid constituents of surfactant are synthesized and secreted by alveolar type II (ATII) pneumocytes (I). However, this cell does not function simply in secretion, since several lines of evidence (2-4) indicate that the constituents of surfactant are taken up by the ATII cells and then reutilized. The presence of a receptor for surfactant protein A (SP-A) on the surface of the cells (5, 6) suggests that receptor-mediated endocytosis (7, 8) may be responsible for the uptake or that SP-A may regulate the secretion of surfactant (9). The intracellular events that result in the secretion of surfactant taken up by the cells remain to be established. Evi(Received in original form May 4, 1990 and in final form January 16, 1991) Address correspondence to: Kevin F. Gibson, M.D., Division of Pulmonary/Critical Care Medicine, Department of Medicine, University of Pittsburgh, School of Medicine, Pittsburgh, PA 15261. Abbreviations: alveolar type II, ATII; Engelbreth-Holm-Swarm tumor, EHS; endoplasmic reticulum, ER; fetal calf serum, FCS; Hepes-buffered saline + 10% FCS, HBS/FCS; phospholipid-to-protein ratio, Pl/Pr ratio; sodium dodecyl sulfate polyacrylamide gel electrophoresis, SDS-PAGE; surfactant protein A, SP-A. Am. J. Respir. Cell Mol. BioI. Vol. 4. pp. 504-513, 1991
dence obtained in studies of the intact lung indicate that the phospholipids of surfactant may recycle without being degraded (2-4, 10-14), while studies on cells in culture suggest that the phospholipids may be degraded and then resynthesized from the degradation products (15). The fate of the apoprotein moiety of surfactant has not been determined. Surfactant is stored within the ATII cells in lamellar bodies, which contain a multilamellar form of surfactant within a membrane-bound structure. How this secretory organelle is formed is not well understood, but it is likely that the surfactant phospholipids and apoproteins are synthesized separately in the endoplasmic reticulum (ER) where they are packaged into transport vesicles. The apoproteins undergo post-translational modification in the ER and Golgi before fusing with phospholipid-containing vesicles to form the mature lamellar body. This organelle may represent the functional equivalent of a storage granule in cells that exhibit regulated secretion of protein (16). In addition, lamellar bodies exhibit properties that are associated with organelles of the endocytic pathway in that they have been shown to contain lysosomal enzymes (17-21) and to fuse with multivesicular bodies (11, 12).
Gibson and Widnell: Lamellar Bodies and Lysosomes in Type II Pneumocytes
During the past few years, considerable progress has been made in characterizing the organelles of the endocytic pathway (22). Endocytic markers are transported first to early endosomes, where ligand-receptor complexes frequently dissociate; they then move to late endosomes, structures that frequently exhibit a multivesicular morphology and that contain lysosomal constituents at a lower concentration than that observed in lysosomes; and they are finally transported to mature lysosomes. If surfactant constituents are recycled without being degraded, they must be diverted from the endocytic pathway to the secretory pathway before degradation can occur. As a first step in analyzing the relationship between the endocytic and secretory pathways in ATII cells, we have characterized the organelles that contain SP-A, and a lysosomal glycoprotein (lgp-120) that is concentrated in lysosomes and also found in late endosomes (22-25). We show here that ATII cells synthesize this glycoprotein in vitro, and that it localizes to a lysosomal compartment. By isopycnic density gradient analysis, we show that two populations of organelles have high phospholipid-to-protein ratios and contain SP-A. The lighter population contains lamellar bodies that lack lysosomal constituents, whereas the denser population consists of multivesicular bodies, lysosomes, and other organelles. The techniques developed in this work should allow the intracellular fate of surfactant to be analyzed during recycling.
Materials and Methods Animals and Reagents Specific pathogen-free male Sprague-Dawley rats (250 to 275 g) were obtained from Zivic-Miller Laboratories and stored in a laminar flow hood prior to use. Ketamine HCI (100 mglml) was obtained from Parke-Davis (Morris Plains, NJ). Percoll, aprotinin, EDTA, N-ethylmalimide, Triton X-114, and fetal calf serum (FCS) were purchased from Sigma Chemical Co. (St. Louis, MO). Electrophoresis reagents were purchased from Bio-Rad (Richmond, CA). Sheep anti-rabbit IgG and fluorescein goat anti-rabbit IgG were obtained from Jackson Immunoresearch (West Grove, PA). Dulbecco's modified Eagle's medium was obtained from GIBCO (Grand Island, NY) and (3sS]methionine from Amersham (Arlington Heights, IL). Engelbreth-HolmSwarm tumor (EHS) basement membrane (Matrigel) was purchased from Collaborative Research (Bedford, MA) and used according to the manufacturer's specifications. 1251 was purchased from New England Nuclear (Boston, MA). A polyclonal antibody directed against rat SP-Awas a generous gift of G. Singh and S. Katyal. A monoclonal antibody directed against a 120-kD glycoprotein (anti-Igp-120) was a generous gift of I. Mellman. Cell Isolation and Culture Type II cell isolation was performed essentially as described by Weller and Karnovsky (26). This procedure yielded 2 x 107 cells/rat, 95 % of which were viable by trypan blue dye exclusion, and approximately 95 % of which were type II cells (confirmed by alkaline phosphatase staining [27], morphology, and the absence of indirect immunofluorescence with mouse monoclonal anti-rat leukocyte common antigen [26]). The plating efficiency averaged ""35%. Cells used in
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metabolic studies were plated on 35-mm petri dishes (coated 30 min earlier with 200 p.l of EHS basement membrane) in Dulbecco's modified Eagle's medium + 10% FCS + antibiotics at a density of 1 x lQ6 cells/mm-. Indirect immunofluorescent studies were conducted on cells cultured on Labtek slides precoated with 50 p.l of EHS basement membrane per chamber (Miles Scientific, Naperville, IL). Cells were stained for alkaline phosphatase activity using an alkaline phosphatase staining kit (Sigma) and were routinely cultured at 1 x 105 cells/mm-. Indirect Immunofluorescence Cells were cultured for 3 d with or without EHS basement membrane prior to staining. Cells were fixed in 3.7% formaldehyde in phosphate-buffered saline at room temperature for 15 min and permeabilized with acetone at -150 C for 2 min. The cells were incubated for 1 h sequentially with Hepesbuffered saline + 10% heat inactivated FCS (HBS/FCS), rabbit anti-rat surfactant protein anti-serum (anti-SP-A) (10 p.l/ml)(28), anti-Igp-120 (1:100 dilution), rat anti-mouse IgG preabsorbed with rabbit serum (50 p.glml) , rhodamine mouse anti-rat IgG (50 p.glml) , and fluorescein-conjugated goat anti-rabbit IgG (50 p.g/ml), with three washes of HBS/FCS between steps. No significant differences were noted if the order of incubation of the fluorescent conjugate antibodies were changed, and no fluorescence was detected at identical exposures if the primary antibodies were omitted. The cells w~re mounted and photographed with an Olympus BH-2 epifluorescence photomicroscope equipped with filters to visualize fluorescein and rhodamine fluorescence. Cell Fractionation Analytical cell fractionation was conducted according to the methods of Tulkens and associates (29). Freshly isolated type II pneumocytes (2 to 3 X 108 cells) were suspended in 0.5 ml of 0.25 M sucrose containing 5 mM Hepes + 0.1 mM EDTA at 4 0 C The cell suspension was then homogenized with 10 strQ~es using a Dounce homogenizer and a B-type pestle, centnfuged at 500 X g for 10 min at 4 0 C, and the supernatant removed. The pellet was rehomogenized in 0.5 ml of the same medium, and the procedure was repeated until a postnuclear supernatant of 2.5 ml was obtained. Low temperature, calcium chelators, and a gentle disruption procedure ensured that lysosomal disruption was minimized during the homogenization procedure. When lysosomal disruption occurred (as was the case in earlier experiments), the enzyme markers were found in both heavy fractions and in at the top of the gradient with other soluble proteins. After removing 0.5 ml to measure homogenate activities, 2.0 ml o~ the postnuclear supernatant (approximately 25 mg of protem) was loaded on a 0.8 to 2.0 M sucrose linear gradient and centrifuged at 100,000 x g for 4 h at 4 0 C in a VTi 50 rotor (Beckman). In experiments using Percoll density gradients, the postnuclear supernatant was brought to a concentr~tion of 10% Percoll to loading on a 25% Percoll gradient WIth a 2.0 M sucrose cushion. The gradients were centrifuged at 70,000 X g for 30 min at 4 0 C in a VTi 50 rotor. Fractions were collected at 4 0 C with a Model 640 fracti?n collector (ISCO) and assayed for N-acetyl-fj-glucosamimdase (30), 5'-nucleotidase (31), succinate dehydrogenase
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(30), NADPH-cytochrome c reductase (32), galactosyl transferase (33), phospholipid-to-protein (PlIPr) ratios (see below), and refractive index (refractometer; American Optical Corporation). Enzyme assays were always carried out under conditions where product formation was linear with respect to time and the amount of cell fraction added to the assay. Enzyme activity recovered in subcellular fractions ranged from 85 to 100 % when compared to the homogenate enzyme activity. Phospholipids were extracted according to the methods of Folch and colleagues (34), and the phospholipid concentration measured according to the methods of Chen and coworkers (35) using egg lecithin as a standard. Protein was determined by the method of Lowry and associates (36). Values reported represent the means of two separate determinations. PlIPr ratios are not reported for fractions obtained on Percoll gradients because of background interference by Percoll. Fractions (""20 % of the total collected) to be analyzed for the presence of Igp-120 and SP-A (200 p,l) were first suspended in a 1:1 dilution of a solution containing 1% Triton X-114 + 150 mM NaCI + 10 mM Tris + aprotinin (0.23 U/ml) + 0.1 mM phenylmethylsulfonyl fluoride + 0.1 mM EDTA at 4 0 C and then vacuum-blotted (Slot blot; Hoefer Scientific Instruments) onto nitrocellulose. (The additional antiproteases were added to ensure that no protein degradation occurred after the vesicles were disrupted with Triton.) After incubation with HBS/FCS for 30 min, the membranes were incubated sequentially with rabbit anti-SP-A (1 p,lIml) for 1 h (to analyze SP-A), or anti-lgp-120 (1:1,000 dilution) and rabbit anti-mouse (1 p,g/ml) for Igp-120 at room temperature. The membranes were rinsed with HBS + 0.005 % Tween" 80 after each antibody incubation. The membranes were then incubated with 12S1 sheep anti-rabbit IgG (5 X lOs cpm/ml), visualized with autoradiography, and quantitated with scanning densitometry (GS 300 scanning densitometer; Hoefer). Densitometry results were linear with respect to the amount of antigen blotted and were expressed as a percentage of the homogenate activity. Electron Microscopy Pooled fractions obtained by analytical cell fractionation of freshly isolated ATII cells were fixed in suspension with 2.5% glutaraldehyde in cacodylate buffer (0.1 M cacodylate + 5 mM MgCl z + 1 mM CaCl z + 2 % sucrose) at 4 0 C for 1 h. Specimens were centrifuged at 100,000 X g for 1 h using an SW50 rotor (Beckman) and washed several times with buffer. The pellets were postfixed in 1% osmium tetroxide in cacodylate buffer, dehydrated with graded alcohol, and embedded in epon. Thin sections were stained with lead citrate and uranyl acetate and photographed with a Jeol JEM 100CX transmission electron microscope. Immunoprecipitation and Electrophoresis Cells cultured on 35-mm dishes were incubated with medium containing (3sS]methionine (100 p,Ci/ml) + 3 % FCS for 3 h at 37 0 C in 5 % CO z• The cells were then washed several times with fresh medium for 1 h. The cells were harvested with dispase (Collaborative Research) according to the manufacturer's instructions. Immunoprecipitation of Igp-120 from Triton X-114 cell homogenates was
performed according to the methods of Lewis and colleagues (23). Immunoprecipitated proteins were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE) (37) using 7.5 % gels and visualized by fluorography (AMPLIFY; Amersham). Immunoprecipitation of SP-A was conducted in a similar fashion using Triton X-100 instead of Triton X-114 and analyzed by SDS-PAGE using 10 to 15% gradient gels. In some experiments, trichloroacetic acid precipitates of cell fractions were evaluated by SDS-PAGE on 7.5 to 15% gradient gels under reducing conditions according to the method of Laemmli (37). Bovine serum albumin (100 p,glml) was added as a carrier prior to acid precipitation. After electroblotting onto nitrocellulose, SP-A was labeled with rabbit anti-SP-A and 1zS1 sheep anti-rabbit IgG and visualized with autoradiography as described above. Other Methods Sheep anti-rabbit IgG was iodinated according to methods of Fraker and Speck (38) and was affinity-purified on a rabbit IgG-Sepharose column prior to use.
Results Type II Pneumocyte Isolation and Culture For these experiments, we selected an isolation procedure that employs both a density gradient centrifugation step and a panning technique in order to obtain a highly purified cell preparation (26). In our first experiments, we studied the behavior of cells isolated by this technique in culture. Cells cultured on EHS basement membrane were rounded in appearance (Figure la) and stained positive for alkaline phosphatase (Figure lc), an enzyme found in high concentration in the plasma membrane of differentiated ATII cells (27). The cells formed aggregates early in culture, but after 3 d, the apical surfaces were still directed toward the overlying medium. After 7 d in culture, the morphologic features of each cell were less distinct as the cell aggregates enlarged, forming cystlike structures as they embedded in basement membrane (not shown). This has been previously described and occurs with other epithelial cells cultured on an EHS basement membrane matrix for extended periods (39,40). The formation of these cystlike structures was not detected before 3 d in our cell preparations. When cultured on plastic, ATII cells rapidly spread and adopted the appearance of squamous cells (Figure lb), The intensity of alkaline phosphatase staining decreased progressively and was scarcely detected at 3 d (Figure 1d). Only an occasional rounded cell could be identified that stained positively for this enzyme. As they dedifferentiated, the nuclei and nucleoli of the cells became more prominent and the large cytoplasmic organelles that conferred a granular appearance to freshly isolated cells gradually disappeared. Synthesis of SP-A and Igp-120 In Vitro Cells cultured on EHS basement membrane were studied to determine if they synthesized surfactant apoproteins and lgp120. After the third day in culture, they were radiolabeled with (3sS]methionine, lysed with a detergent buffer, immunoprecipitated with anti-lgp-120 and anti-SP-A, and analyzed as described in MATERIALS AND METHODS. As Figure
Gibson and Widnell: Lamellar Bodies and Lysosomes in Type II Pneumocytes
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c
Figure 1. Type ITpneumocytes were cultured for 3 d on Engelbreth-Holm-Swarm tumor (EHS) basement membrane (a and b) or tissue culture grade plastic (c and d), fixed, and stained according to methods described in MATERIALS AND METHODS. (a and c) Nomarski differential interference contrast microscopy; (b and d) phase-contrast images after alkaline phosphatase staining. Cells cultured on EHS basement membrane were more rounded in appearance and stained positively for alkaline phosphatase. When cultured on plastic, the cells rapidly spread, adopting the appearance of squamous cells. The intensity of staining of alkaline phosphatase decreased progressively in these cells, and most cells showed no detectable activity (bar = 16 Itm).
2 demonstrates, both SP-A and lysosomal integral membrane glycoprotein 19p-120 are synthesized by type II pneumocytes in culture. Anti-SP-A precipitated a heterogeneous group of proteins ranging in molecular mass between 26 and 36 kD, representing varying degrees of post-translational glycosylation of the protein (28, 41). The most prominent bands were at 32 and 38 kD. The heavily sialated lysosomal glycoprotein migrated as a broad band with an average M, of 120 kD, when analyzed under reducing conditions (23). Indirect Immunofluorescence Studies of SP-A and Igp-120 SP-A in cells cultured on EHS basement showed a diffuse dense pattern of immunofluorescence similar in appearance . to cells stained in situ (Figure 3a) (41). Staining patterns typical of individual organelles were hard to detect in these cells because their cuboidal shape caused the images of organelles to become superimposed, limiting their resolution at the light-microscope level. The pattern of fluorescence of 19p120 (Figure 3b) appeared more punctate, with some areas that overlapped with SP-A fluorescence; however, there were structures that apparently stained with either anti-SP-A or anti-lgp-120 but not both antibodies. The pattern of staining raised the possibility that there might be two populations of SP-A-containing structures (presumed lamellar bodies), one of which contained 19p-120 while the other lacked the lysosomal glycoprotein.
A Figure 2. Synthesis of surfactant protein A (SP-A) and Igp-120. Type II pneumocytes cultured for 3 d on EHS basement membrane were radiolabeled with [35S]methionine. Immunoprecipitates of SP-A (A) and Igp-120 (B) from detergent cell lysates were analyzed according to the procedure described in MATERIALS AND METHODS.
B
In order to better resolve organelles at the lightmicroscope level, the localization of SP-A and 19p-120 was investigated in cells cultured on tissue culture grade plastic (Figures 3c and 3d). These cells were much more spread out. When double-stained with the two antibodies and studied by fluorescence microscopy, large vesicular structures in the perinuclear region stained with both antibodies. All structures that stained positively for 19p-120 (Figure 3d) also stained for SP-A (Figure 3c). In addition, there was a very fine, and somewhat hard to detect, punctate immunofluorescence in cells stained for SP-A (Figure 3c), which did not appear to localize to the plasma membrane (i.e., not in the plane of focus of the plasma membrane) and did not label with Igp-120. Analytical Fractionation of Type II Pneumocytes The intracellular location of SP-A and Igp-120 was also investigated by analytical cell fractionation. Freshly isolated cells were studied to avoid any possible redistribution of markers that might occur during culture. Cell homogenates were fractionated on continuous linear sucrose gradients, and fractions were analyzed for PI/Pr ratios, 5'-nucleotidase, N-acetyl-J3-glucosaminidase, succinate dehydrogenase, NADPH-cytochrome c reductase, galactosyl transferase, SP-A, and Igp-120. The distribution of phospholipid and protein is shown in Figure 4a. There were two populations of organelles that contained high PlIPr ratios; one peak appeared at a density of 1.05 (0.55 M sucrose), while the second peak appeared at a density of 1.10 (1.19 M sucrose). The high PlIPr ratios identify structures that contain surfactant phospholipids; the PlIPr ratio of the postnuclear supernatant averaged 4.3 ~mollmg.
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93
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Figures 4b and 4d show the distribution of lysosomal markers. Approximately 84% of the N-acetyl-J3-glucosaminidase activity was recovered at densities ranging between 1.13 and 1.20 (1.03 to 1.58 M sucrose), with a peak activity occurring at a density of 1"\.11.16 (Figure 4a). The lysosomal glycoprotein Igp-120 was recovered in the same fractions that contained N-acetyl-J3-glucosaminidase (Figure 4b). The figures shown are representative graphs obtained from one of four separate experiments that yielded qualitatively simi-
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AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 4 1991
Figure 3. Double indirect immunofluorescent staining of SP-A and Igp-120 in type II pneumocytes cultured on EHS basement membrane (a and b) (bar = 8 /Lm) or tissue grade plastic (c and d) (bar = 16 /Lm). Cells cultured on EHS basement membrane showed a diffuse dense pattern of immunofluorescence when stained for SP-A. The pattern of fluorescence of Igp-120 appeared more punctate, with areas that appeared to overlap with SP-A fluorescence; however, there were some structures that apparently stained with either anti-SP-A or anti-lgp-120 but not both antibodies. In cells cultured on plastic, large vesicular structures in the perinuclear region stained with both antibodies (arrows). In addition, there was a very fine and hard to detect punctate immunofluorescence in cells stained for SP-A which was not in the plane of focus of the plasma membrane and did not label with Igp-120.
lar results. The findings are consistent with previous observations that Igp-120 is localized primarily to late endosomal (prelysosome) and lysosomal membranes (23, 24, 42). In addition, the lighter portion of the lysosomal fraction appeared to overlap with the second PI/Pr peak, consistent with the presence of surfactant phospholipids within these structures. However, the light PI/Pr peak lacked lysosomal markers. The amount of SP-A (Figure 4c) was also assayed in each fraction , and the distribution showed two distinct peaks that were associated with high PI/Pr ratios. One peak occurred at a density of 1.05 (0.55 M sucrose) in a region devoid of N-acetyl-{3-glucosaminidase and Igp-120 and represented a region of the gradient where lamellar bodies are reported to migrate (43). In addition, there was a second peak at a density of 1.11 to 1.20 that co-localized with Igp-120and N-acetyl-{3glucosaminidase, suggesting that a population of vesicles bearing lysosomal markers (lgp-120 and N-acetyl-{3-glucosaminidase) and an elevated PI/Pr ratio, also contained SP-A. The percentage of SP-A recovered in fractions containing lysosomal markers varied from N60 to 90% of the total SP-A recovered in each experiment. The distribution of 5'-nucleotidase (plasma membrane), NADPH-cytochrome c reductase (ER), succinate dehydrogenase (mitochondria), and galactosyl transferase (Golgi) was also assayed, and the results are shown in Figure 4. 5'-nucleotidase (Figure 4e) and galactosyl transferase (Figure 4g) were detected in fractions with densities of 1.10 to 1.15, a region that partially overlaps with the lysosomal markers. NADPH-cytochrome c reductase (Figure 4h) and succinate dehydrogenase (Figure 4f) activities appeared in denser fractions, with density ranging between 1.113 and 1.20. None of these markers coincided with the light fraction that contained SP-A and a high PI/Pr ratio. Fractions from the lamellar body peak (labeled I in Figure 4) and the light (II) and dense (II) lysosomal peaks were pooled and studied by electron microscopy. Most of the structures in pooled fraction I consisted of lamellar bodies (Figure Sa). Many were surrounded by a distinct membrane and apparently intact (Figure 5b). Others, however, appeared partly disrupted-perhaps during preparation for
electron microscopy, and there were also smooth vesicles in the fraction (Figure Sa). The constituents of pooled fraction II are shown at low magnification in Figure 6a. This fraction contained vesicles, multivesicular bodies, lamellar bodies, mitochondria, Golgi stacks, and lysosomes. The multivesicular bodies were packed with vesicles and, in some instances, clearly identifiable lamellae (Figure 6b). Some contained vesicles and lamellae in an electron-dense matrix characteristic of lysosomes (Figure 6c). Pooled fraction ill (Figure 7) contained mostly dense lysosomes and mitochondria. In order to determine if the SP-A associated with the dense fractions was undergoing degradation, trichloroacetic acid precipitates of the fractions were analyzed by Western blot. As is shown in Figure 8, most of the SP-A detected in fractions with a density of 1.088 to 1.100 appeared to be intact protein (Figure 8b, I). However, in the more dense fractions (1.115 to 1.185), there was a smear of low-molecularweight proteins that were labeled by the antibody, suggesting that SP-A is undergoing degradation in these fractions (Figure 8b, 2 and 3). The amount of degraded apoprotein comprised a significant percentage of the total protein detected on the immunoblot and suggests that the apoprotein is present in, and being degraded by, Iysosomes. Analytical cell fractionation was carried out using Percoll density gradients in order to better resolve structures in the high-density regions . As Figure 9 demonstrates, two populations of vesicles that contained N-acetyl-{3-glucosaminidase and Igp-120 were detected at densities of Nl.l0 and N1.35. In addition, SP-A was associated with three distinct peaks (Figure 9c); only the second and third peaks contained 19p120 and N-acetyl-{3-glucosaminidase (Figures 9b and 9c).
Discussion The purpose of this study was to examine the role of the lysosome in the recycling of surfactant by ATII cells. We studied the distribution of SP-A in ATII cells in culture by immunofluorescence; cells cultured on EHS basement membrane showed SP-A in a diffuse staining pattern with areas that did not co-localize with Igp-120. In contrast, most of the
Gibson and Widnell: Lamellar Bodies and Lysosomes in Type II Pneumocytes
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