0022-1554/92/$3.30 The Journal of Histochemistry and Cytochemistry Copyright 0 1992 by The Histochemical Society. Inc
Vol. 40, No. 10, pp. 1589-1597, 1992 Printed in US.A.
Original Article
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Immunocytochemical Localization of Surfactant Protein D (SPD) in Type I1 Cells, Clara Cells, and Alveolar Macrophages of Rat Lung WIM E VOORHOUT,' TINEKE VEENENDAAL, YOSHIO KUROKI, YOSHINORI OGASAWARA, LAMBERT M. G. VAN GOLDE, and HANS J. GEUZE Department of Cell Biology, Medical School m W H J G ) ) ,and Laboratory of Vaterinary Biochemistry m L M G G ) , University of Utrecht, Utrecht, The Netherlands, and Department of Biochemistry, Sapporo Medical College (YK,YO), Sapporo, Japan. Received for publication January 3, 1992 and in revised form May 11, 1992; accepted May 23, 1992 (2A2560).
We investigated the cellular and subcellular distribution of surfactantprotein D (SPD)by immunogold l a w g in lungs of adult rats that had been given bovine"es albumin coupled to 5-nm gold (BSAG) for 2 hr to visualize the endocytotic pathway. Specific gold labeling for SPD was found in alveolar Type II cells, Clara cells, and alveolar macrophages. In Type II cells abundant labeling was observed in the endoplasmic reticulum, whereas the Golgi complex and multivesicular bodies were labeled to a limited extent only. Lamellar bodies did not seem to contain SPD. Gold labeling in alveolar macrophageswas restricted to structurescontaining endocytosed BSAG. In Clara c e h labeling was found in the endoplasmic reticulum, the Golgi complex, and was most
,ntroduction Pulmonary surfactant is a complex mixture of lipids and proteins (1,2) which prevents the alveoli from collapsing at end expiration (3). Surfactant is synthesized by alveolar Type I1 cells, in which it is stored in lamellar bodies. On fusion of the limiting membrane of lamellar bodies with the apical plasma membrane, pulmonary surfactant is secreted into the alveolar space, where it is transformed into tubular myelin (4). Some surfactant constituents are not synthesized solely by the alveolar Type I1 cell but also by non-ciliated broncheolar epithelial (Clara) cells (5,6). Surfactant protein D (SP-D)is a collagenous surfactant-associated glycoprotein which is synthesized and secreted by freshly isolated rat Type I1 cells and has been isolated from rat bronchoalveolar lavage fluids (7-9). In rat lung it is synthesized as a 39.3 KD primary translation product that undergoes post-translational N-linked glycosylation, as well as hydroxylation and glycosylation of lysine residues within the collagenous domain (10).SP-D is secreted as a 43 KD protein and is isolated from bronchoalveolar lavage as a Correspondence to: Wim E Voorhout, Univ. of Utrecht, Dept. of Cell Biology, Medical School, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands.
prominent in granules present in the apical domain of the cell. Double labeling experimentswith anti-surfactant protein A (SPA) showed that both SPA and SPD were present in the same granules. However, SPA was distributed throughout the granule contents, whereas SPD was confined to the periphery of the granule. The Clara cell granules are considered seaetory granules and not lysosomes, because they were not labeled for the lysosomal markers cathepsin D and LGP120,and they did not contain endocytosed BSAG. ( J Histochem Cytochem 40:1589-1597, 1992) KEY WORDS: Rat lung surfactant; Alveolar type I1 cells; Clara cells; Immunogold labeling; Surfactant protein D; Surfactant protein A.
multimeric complex of disulfide-bonded trimers composed of apparently identical subunits. SP-D is a calcium-dependent carbohydrate binding protein (9) which is structurally related to surfactant protein A (SP-A), conglutinin, and other collagenous C-type lectins (11,12). The structural similarities of SP-D and SP-A suggest that these two proteins have similar functions. Both SP-A (13) and SP-D (14) have been found immunohistochemically in alveolar Type I1 cells and Clara cells of the rat lung at the light microscopic (LM) level, and recently SP-D has been localized in Type I1 cells and in the airspace of silica-treated rats at the electron microscopic (EM) level (15). In this study we localized SP-Din adult rat lung at the EM level and, since SP-D is structurally related to SP-A, compared its distribution with that of SP-A. We show that SP-D is secreted from Clara cells via granules that also contain SP-A. In alveolar Type I1 cells SP-D is probably not secreted via lamellar bodies, the intracellular storage site of pulmonary surfactant, but via a different secretion pathway.
Materials and Methods Immunoreagents. The SP-D antibody was raised in rabbit against purified rat SP-D as described (16)and is an IgG fraction (0.85 mg/ml) puri1589
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fied by a protein A-Sepharose CL 4B column. By immunoblotting analysis the antibody recognizes a single protein of 43 KD in preparations of whole surfactant, bronchoalveolar lavage fluid from normal rat. adult rat alveolar Type I1 cells, and adult rat lung homogenate. It reacts with purified rat SP-D but gives no reaction with purified rat SP-A (16). The SP-A antibody is a purified rabbit IgG polyclonal antibody against rat SP-A (antiserum kindly provided by Dr. S. Hawgood) and reacts only with the three known isoforms of SP-A with molecular masses of 36 KD, 32 KD, and 28 KD by immunoblot analysis of isolated rat alveolar Type I1 cells (17). The antibody against cathepsin D was a gift of Dr. K. von Figura (18) and the antiserum against LGPl20 was a rabbit serum against rat lysosomal membrane protein, kindly provided by Dr. I. Mellman (19). Bovine serum albumin coupled to 5-nm gold particles (BSAG) and protein A-gold complexes(PAG) of different size classes were prepared by the tannic acid-citrate method ) mixed with (20). For endocytosis studies, 475 pl BSAG (OD520 ~ 7 5were 25 pI of isolated rat surfactant (corresponding to 10% of total amount of surfactant isolated from one rat lung lavage). Surfactant was isolated as described before (21). This fluid-phase endocytosis mixture was dialyzed overnight at 4°C against Gey's balanced salt solution containing 10 mM Hepes, pH 7.4.
Cryoultramicrotomyand Immunogold Labeling. Rat lungs were given an intratracheal instillation of 500 pI fluid-phase endocytosismixture (BSAG + surfactant) to mark the endocytic pathway in lung epithelial cells. After 2 hr the rats were anesthetized with sodium pentobarbital (30 mglkg body weight), their chests were opened, and lungs were fixed by perfusion via the right ventricle with 2 % paraformaldehyde, 0.2% glutaraldehyde, 1%
2
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Figure 1. Ultra-thin cryosectionof rat lung showing tubular myelin in the alveolar space that is devoid of labeling for SP-D. Section was labeled with anti-SP-0 and PAG (20 nm). Bar = 0.2 pm.
Figure 2. Rat lung alveolar macrophage that has endocytosed BSAG (5 nm) for 2 hr before fixation. Section was labeled with anti-SP-0 and PAG (10 nm). Endosomal and lysosomal structures are filled with BSAG and contain some SP-D (arrows). Bar = 0.2 pm.
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LOCALIZATION OF SURFACTANT PROTEIN D
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Figure 3. lmmunogold labeling of SP-D in rat lung alveolar Type II cells with (A) 10-nm PAG and (B) 20-nm PAG. The small gold represents endocytosed BSAG (5 nm). The endoplasmic reticulum (er) (A) and the Golgi complex (9) (e) are clearly labeled, whereas lamellar bodies (Ib) were negative. Bars = 0.2 pm.
acrolein, 1.25% dimethylsulfoxide in 0.1 M phosphate buffer. p H 7.4. The lungs were removed and further fixed by immersion in the same fixative. The tissue was cut into thin slices and infiltrated with increasing concentrations of gelatin up to 10% (vlv) in 0.1 M phosphate buffer. p H 7.4. at 37'C. After solidification of the gelatin at 4°C. small blocks of approximately 1 mm3 were cut and infiltrated with 2.3 M sucrose overnight at 4°C and frozen in liquid nitrogen. Ultra-thin cryosections were cut on a Reichert UltracutEIFC4E (Leica
Aktiengesellschaft; Wien. Austria) at - 100°C with a 45' cryodiamond knife (Diatome; Bienne, Switzerland) and transferred to formvar-carboncoated copper grids. Thawed ultra-thin cryosections were floated on 2% gelatin in PBS at 37°Cfor 10 min to block nonspecific binding sites, on 0.02% glycinelPBS for 10 min to block remaining free aldehyde groups. and subsequently on the primary antibody against SP-D diluted 1:50 in 1% BSAlPBS for 30-60 min. Sections were rinsed three times for 5 min with 1% BSAlPBS and immunoreactivity was visualized by incubation with
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Figure 4. lmmunogold labeling of SP-D in a rat lung Clara cell with 20-nm PAG. The lung tissue was allowed to endocytose 5-nm BSAG for 2 hr before fixation. (A) Overview of the apical part of a Clara cell with prominent labeling at the rims of secretory granules (sg). A ciliated epithelial cell (CE) is devoid of intracellular labeling. (B) Golgi complex (9) located close to the nucleus (n) which is clearly labeledfor SP-D. (C) Granule close to the nucleus (n) with a matrix density comparable to that of the cytoplasm, showing labeling for SP-D at the rim of the granule. (D) Electron-dense secretory granule near the apical plasma membrane. SP-D is clearly located at the rim of the electron dense matrix. Bars: A = 1 pm; B-D = 0.2 km.
PAC for 30 min. In double labeling experiments the last step was followed by a short rinse with PBS, fixation of the sections with 1% glutaraldehyde in PBS for 10 min, and blocking with 0,020h glycine in PBS beforc]abc]ing with the second antibody and PAG o f a different size (22). control experiments were performed by omission of the primary antibody or by using an irrelevant antibody (i.e., anti-amylase). Finally, labeled sections were rinsed four times for 5 min with distilled water, stained on a drop of uranyl acetate-oxalate, pH 7, for 5 min. and embedded in 1.8% methylcellulose containing 0.4% uranyl acetate. Sections were observed and photographed in a Jeol l2OOEX electron microscope at 80 kV.
Results I' In the rat lung Only and *macrophageswere specifically labeled with the antibody against SP-D. Alveolar Type I cells, interstitial cells, and ciliated bronchiolar epithelial cells showed no labeling for SP-D. Control experiments in which the primary antibody was omitted or substituted with an irrelevant antibody (anti-mylase)showed negligible goid labeling (data not shown). Tubular myelin, one ofthe extracellularforms of surfactant, contained no detectable SP-D (Figure 1). In alveolar
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macrophages SP-D occurred in endocytotic and lysosomal structures (Figure 2) showing endocytosed BSAG, but was absent from the ER and the Golgi complex. This suggests that intracellular SP-D derives from uptake of SP-D from the alveolar subphase. However, in both Type I1 cells and Clara cells the biosynthetic organelles were labeled (Figures 3 and 4). In Type I1 cells SP-D was found within the lumen of the ER cisternae and in the Golgi complex (Figure 3). Lamellar bodies, the intracellular storage compartment of surfactant, were almost completely devoid of labeling (Figure 3). Only 10% of the lamellar bodies (n = 113) contained a few gold particles. In addition, in sections double labeled for SP-A and SP-D both surfactant proteins co-localized in the ER and the Golgi complex of alveolar Type I1 cells, whereas lamellar bodies contained only SP-A and no SP-D (data not shown). Multivesicular bodies (MVB), which are thought to be part of the biosynthetic pathway of surfactant proteins (23). were also labeled to a limited extent, since only 20% of the MVB (n = 30) contained detectable amounts of SP-D. Although Type I1 cells were clearly labeled, the most prominent labeling was found in the Clara cells of the respiratory bronchioles (Figure 4A). Both the ER and the Golgi complex were sparsely but specifically labeled (Figure 4B). Golgi complexes were located primarily at the apical and basolateral sides of the nucleus, which in turn was situated in the basal part of the Clara cell. At the apical site of the nucleus large granules (0.2-1 pm in diameter), with a variable matrix density and surrounded by a single membrane, were profoundly labeled (Figures 4A. 4C, and 4D). The matrix density of these granules varied from a density comparable to that of the cytoplasm (Figure 4C) to very electron dense, sometimes with a crystalline appearance (Figures 4D and 8). The distribution of SP-D in the Clara cell granules was heterogeneous. SP-D labeling was found predominantly in between the granule content and the limiting membrane (Figures 4A and 4D). Neither in granules with a very dense matrix nor in granules with a loosely packed matrix was a significant amount of label found in the matrix. Most of the granules with a loosely packed matrix were found close to the nucleus, whereas granules at the upper apical part of the cell had a much more dense matrix. Occasionally exocytotic profiles of granules were encountered (Figure 5). To discriminate between the biosynthetic and the endocytotic compartments, the lung epithelial cells were allowed to endocytose BSAG continuously for 2 hr. BSAG was found in small vesicles, in MVB, and in lysosomes. The vast majority of the SP-Dcontaining granules contained no endocytosed BSAG (Figures 4-7) and did not label for the lysosomal markers LGPl20 (Figure 6) and cathepsin D (Figure 7). Occasionally a dense granule, exhibiting morphological resemblance to dense SP-D-containinggranules, contained endocytosed BSAG (Figures 6 and 7). Some of these BSAGpositive granules also contained SP-D (Figure 6). These BSAGcontaining dense granules, which occurred predominantly in the basal part of the cells, are lysosomes, since they contained LGPl20 (Figure 6) and cathepsin D (Figure 7). From previous studies it is known that the other hydrophilic surfactant protein, SP-A. is found in dense granules of rat lung Clara cells (13). In double labeling experiments it was clear that SP-A and SP-D were present in the same granules (Figures 8B8D), but within these granules SP-A and SP-D displayed different distributions.
Figure 5. Exocytotic profile of a secretory granule in a Clara cell labeled for SP-D with 20-nm PAG. Bar = 0.2 vm.
Whereas most SP-D was present at the periphery of the granule, in between the matrix and the limiting membrane, SP-A was homogeneously distributed over the matrix of the granule, indicating a spatial separation between the two surfactant proteins (Figures 8B-8D). In the biosynthetic organelles, i.e., the Golgi complex, SP-A and SP-D co-localized, whereas the labeling densities suggested that much more SP-A is present than SP-D (Figure 8A).
Discussion Pulmonary surfactant is a complex mixture of lipids and proteins (1,2). At present, four classes of proteins are considered to be specific surfactant proteins (24). These surfactant proteins are referred to as SP-A. SP-B, SP-C and SP-D according to the nomenclature proposed by Possmayer (25). The hydrophilic surfactant protein SP-A has been extensively localized at the EM level (13J7.26-28), and these studies have shown that SP-A is restricted to alveolar Type I1 cells, alveolar macrophages, tubular myelin, and Clara cells of the rat lung. In this study we have localized the structurally related SP-D using high-resolution immuno-EM. SP-D was present in alveolar Type I1 cells, alveolar macrophages, and Clara cells of the rat lung, but was absent from tubular myelin, which is in agreement with previous localization studies at the LM level (14) and the EM level (15). Although both SP-A and SP-D are synthesized and secreted by Type I1 cells, their subcellular distributions were found to differ. Whereas both proteins are present in the ER and the Golgi complex, lamellar bodies do not seem to contain SP-D in contrast to SP-A, which is present in all lamellar bodies (17). In addition, tubular myelin, one of the extracellular forms of surfactant originating from secreting lamellar bodies (4), is not labeled for SP-D. However. in a previous study we have shown that tubular myelin is heavily la-
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VOORHOUT, VEENENDAAL. KUROKI, OGASAWARA, VAN GOLDE, GEUZE
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Figures 6 and 7. Ultra-thincryosections of Clara cells double labeled for SP-D with 15-nm PAG and the lysosomal membrane glycoprotein LGPl2O with IO-nm PAG (Figure 6) or the soluble lysosomal enzyme cathepsin D (CDA) with 10-nm PAG (Figure 7). The lung tissue was allowed to endocytose 5-nm ESAG for 2 hr before fixation. Secretory granules (sg) do not contain endocytosed ESAG and are not labeled for LGPlPO (Figure 6) or cathepsin D (Figure 7), whereas ESAG which are morphologically similar to the secretory granules, are labeled for LGPlPO (Figure 6) and cathepsin D (Figure 7) and some(arrow)-positivegranules times also contain SP-D (Figure 6). Ears = 0.5 pm.
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Figure 8. Rat lung Clara cells, fixed after 2 hr of uptake of 5 n m BSAG, double labeled for SP-D (15-nm PAG) and SP-A (10-nm PAG). (A) The Golgi complex (9) is abundantly labeled for SP-A and to a lesser extent for SP-D. (B-D) Secretory granules are homogeneously labeled for SP-A, whereas SP-D is mostly located at the rims of the granules. (D) Some granules show a crystalline matrix with the same distribution of SP-A and SP-D. Bars: A-C = 0.2 Km; D = 0.1 pm.
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beled for SP-A (17). In addition, recent studies have shown that extracellular SP-D distributes differently from SP-A. Crouch et al. (15) showed that in silica-treated rats SP-D is associated with granular material but not with tubular myelin. In alveolar lavage fluids, Kuroki et al. (29) showed that 70% of SP-D was present in the 33,000 x g supernatant, whereas about 99% of SP-A was associated with the 33,000 x g pellet, suggesting that most SP-D, although associated with lipids, is not incorporated into a large lipid-protein complex. These data, together with our localization studies, suggest that SP-D is not secreted together with the lipid-protein complex via lamellar bodies but via another secretory pathway. Since SP-A and SP-D co-localize in the Golgi complex, the diversion of SP-Dfrom the other surfactant proteins must take place somewhere between the Golgi complex and the lamellar bodies. The precise location at which SP-D is sorted from the other surfactant proteins remains to be elucidated. In the Clara cell of the rat lung, SP-D is present in the biosynthetic organelles, i.e., in the ER and the Golgi complex, and in immature and mature secretory organelles. The immature granules in the Golgi area, exhibiting a moderately dense content, seem to give rise to apically located mature secretory granules with a dense content, sometimeseven crystalline. In double labeling experiments both SP-D and SP-A were localized in the same secretory granule and are therefore secreted together via the same organelle, in contrast to the apparently different secretory routes of SP-A and SP-D in Type I1 cells. Although SP-A and SP-D were present in the same granule, they were clearly separated from each other within the same granule. The localization of SP-D at the periphery of the dense granules might be due to the exclusion of SP-D from the condensing matrix. SP-D might also have an affinity with the limiting membrane, since even in early granules SP-D is localized at the periphery of these granules. From this study and previous studies, it is evident that there are at least two secretory organelles of distinct nature that are involved in the secretion of surfactant components. Alveolar Type I1 cells have a lysosome type of secretory organelle, the lamellar body, which is connected to the endocytotic pathway. Lamellar bodies secrete the surfactant lipids together with the surfactant proteins SP-A, SP-B,and SP-C. Our immuno-EM observations suggest that lamellar bodies are devoid of SP-D. The other type of secretory organelle is found in Clara cells. These secretory organelles have no lysosomal characteristics, they are not connected to the endocytotic pathway, and they are devoid of large amounts of phospholipids, since tritiated choline is not incorporated into these granules (30). They do secrete the hydrophilic surfactant proteins SP-A and SP-D, but not the hydrophobic surfactant protein SP-B (data not shown), whereas SP-B is abundantly present in lamellar bodies (31,32). A clear function for SP-D is not known, but it seems that SP-D is not important for the structural organization of the surfactant lipids, since it is not associated with the bronchoalveolarlavage pellet (29), it is not secreted together with the surfactant lipids, it is not found in tubular myelin, and it is not able to aggregate phospholipid liposomes (29). SP-A, on the other hand, is an essential component in the structural organizationof surfactant, since it is necessary for the formation of tubular myelin in vitro (33,34), it is localized at the corners of the tubular myelin network (17), and
it is able to aggregate phospholipid liposomes (35,36). Developmental profiles of SP-D expression in rat lung (14,16) also suggest that SP-D is not needed in significant amounts before birth and that it may not be required for the formation of surface active material. In addition to its structural role in surfactant, SP-A may play an important role in the alveolar defense mechanism (21,37,38), and it might well be that SP-D, which is structurally related to SPA, conglutinin, and other C-type lectins (11,12), also has a function in the lung defense mechanism. Such a possible function of SP-D in alveolar defense has also been suggested by others (14,39,40). Since Clara cells secrete both SP-A and SP-D, and no other surfactant components, these cells could play an important role in the host-defense mechanism of the rat lung.
Acknowledgments We are grateful to Or S.Hawgood (University of Ca1;forniaSan Francisco, San Francisco)for his gift of anti SPA, to Dr K. von Figura (Georg August Universitat, Gottingen) for the anti-cathepsin D, to Dr I. Mellman pale University, New Haven) for the anti-LGPl20, and to Drs Toyoaki Akino, Hen&E! Haagsman, andJan K Slot for stimulating discussions andcritically reading the manuscript. We thank Tom van Rqn andMaurits Niekerk for preparing the micrographs.
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Vol. 40, No. 10, pp. 1589-1597, 1992 Printed in US.A.
Original Article
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Immunocytochemical Localization of Surfactant Protein D (SPD) in Type I1 Cells, Clara Cells, and Alveolar Macrophages of Rat Lung WIM E VOORHOUT,' TINEKE VEENENDAAL, YOSHIO KUROKI, YOSHINORI OGASAWARA, LAMBERT M. G. VAN GOLDE, and HANS J. GEUZE Department of Cell Biology, Medical School m W H J G ) ) ,and Laboratory of Vaterinary Biochemistry m L M G G ) , University of Utrecht, Utrecht, The Netherlands, and Department of Biochemistry, Sapporo Medical College (YK,YO), Sapporo, Japan. Received for publication January 3, 1992 and in revised form May 11, 1992; accepted May 23, 1992 (2A2560).
We investigated the cellular and subcellular distribution of surfactantprotein D (SPD)by immunogold l a w g in lungs of adult rats that had been given bovine"es albumin coupled to 5-nm gold (BSAG) for 2 hr to visualize the endocytotic pathway. Specific gold labeling for SPD was found in alveolar Type II cells, Clara cells, and alveolar macrophages. In Type II cells abundant labeling was observed in the endoplasmic reticulum, whereas the Golgi complex and multivesicular bodies were labeled to a limited extent only. Lamellar bodies did not seem to contain SPD. Gold labeling in alveolar macrophageswas restricted to structurescontaining endocytosed BSAG. In Clara c e h labeling was found in the endoplasmic reticulum, the Golgi complex, and was most
,ntroduction Pulmonary surfactant is a complex mixture of lipids and proteins (1,2) which prevents the alveoli from collapsing at end expiration (3). Surfactant is synthesized by alveolar Type I1 cells, in which it is stored in lamellar bodies. On fusion of the limiting membrane of lamellar bodies with the apical plasma membrane, pulmonary surfactant is secreted into the alveolar space, where it is transformed into tubular myelin (4). Some surfactant constituents are not synthesized solely by the alveolar Type I1 cell but also by non-ciliated broncheolar epithelial (Clara) cells (5,6). Surfactant protein D (SP-D)is a collagenous surfactant-associated glycoprotein which is synthesized and secreted by freshly isolated rat Type I1 cells and has been isolated from rat bronchoalveolar lavage fluids (7-9). In rat lung it is synthesized as a 39.3 KD primary translation product that undergoes post-translational N-linked glycosylation, as well as hydroxylation and glycosylation of lysine residues within the collagenous domain (10).SP-D is secreted as a 43 KD protein and is isolated from bronchoalveolar lavage as a Correspondence to: Wim E Voorhout, Univ. of Utrecht, Dept. of Cell Biology, Medical School, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands.
prominent in granules present in the apical domain of the cell. Double labeling experimentswith anti-surfactant protein A (SPA) showed that both SPA and SPD were present in the same granules. However, SPA was distributed throughout the granule contents, whereas SPD was confined to the periphery of the granule. The Clara cell granules are considered seaetory granules and not lysosomes, because they were not labeled for the lysosomal markers cathepsin D and LGP120,and they did not contain endocytosed BSAG. ( J Histochem Cytochem 40:1589-1597, 1992) KEY WORDS: Rat lung surfactant; Alveolar type I1 cells; Clara cells; Immunogold labeling; Surfactant protein D; Surfactant protein A.
multimeric complex of disulfide-bonded trimers composed of apparently identical subunits. SP-D is a calcium-dependent carbohydrate binding protein (9) which is structurally related to surfactant protein A (SP-A), conglutinin, and other collagenous C-type lectins (11,12). The structural similarities of SP-D and SP-A suggest that these two proteins have similar functions. Both SP-A (13) and SP-D (14) have been found immunohistochemically in alveolar Type I1 cells and Clara cells of the rat lung at the light microscopic (LM) level, and recently SP-D has been localized in Type I1 cells and in the airspace of silica-treated rats at the electron microscopic (EM) level (15). In this study we localized SP-Din adult rat lung at the EM level and, since SP-D is structurally related to SP-A, compared its distribution with that of SP-A. We show that SP-D is secreted from Clara cells via granules that also contain SP-A. In alveolar Type I1 cells SP-D is probably not secreted via lamellar bodies, the intracellular storage site of pulmonary surfactant, but via a different secretion pathway.
Materials and Methods Immunoreagents. The SP-D antibody was raised in rabbit against purified rat SP-D as described (16)and is an IgG fraction (0.85 mg/ml) puri1589
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fied by a protein A-Sepharose CL 4B column. By immunoblotting analysis the antibody recognizes a single protein of 43 KD in preparations of whole surfactant, bronchoalveolar lavage fluid from normal rat. adult rat alveolar Type I1 cells, and adult rat lung homogenate. It reacts with purified rat SP-D but gives no reaction with purified rat SP-A (16). The SP-A antibody is a purified rabbit IgG polyclonal antibody against rat SP-A (antiserum kindly provided by Dr. S. Hawgood) and reacts only with the three known isoforms of SP-A with molecular masses of 36 KD, 32 KD, and 28 KD by immunoblot analysis of isolated rat alveolar Type I1 cells (17). The antibody against cathepsin D was a gift of Dr. K. von Figura (18) and the antiserum against LGPl20 was a rabbit serum against rat lysosomal membrane protein, kindly provided by Dr. I. Mellman (19). Bovine serum albumin coupled to 5-nm gold particles (BSAG) and protein A-gold complexes(PAG) of different size classes were prepared by the tannic acid-citrate method ) mixed with (20). For endocytosis studies, 475 pl BSAG (OD520 ~ 7 5were 25 pI of isolated rat surfactant (corresponding to 10% of total amount of surfactant isolated from one rat lung lavage). Surfactant was isolated as described before (21). This fluid-phase endocytosis mixture was dialyzed overnight at 4°C against Gey's balanced salt solution containing 10 mM Hepes, pH 7.4.
Cryoultramicrotomyand Immunogold Labeling. Rat lungs were given an intratracheal instillation of 500 pI fluid-phase endocytosismixture (BSAG + surfactant) to mark the endocytic pathway in lung epithelial cells. After 2 hr the rats were anesthetized with sodium pentobarbital (30 mglkg body weight), their chests were opened, and lungs were fixed by perfusion via the right ventricle with 2 % paraformaldehyde, 0.2% glutaraldehyde, 1%
2
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Figure 1. Ultra-thin cryosectionof rat lung showing tubular myelin in the alveolar space that is devoid of labeling for SP-D. Section was labeled with anti-SP-0 and PAG (20 nm). Bar = 0.2 pm.
Figure 2. Rat lung alveolar macrophage that has endocytosed BSAG (5 nm) for 2 hr before fixation. Section was labeled with anti-SP-0 and PAG (10 nm). Endosomal and lysosomal structures are filled with BSAG and contain some SP-D (arrows). Bar = 0.2 pm.
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Figure 3. lmmunogold labeling of SP-D in rat lung alveolar Type II cells with (A) 10-nm PAG and (B) 20-nm PAG. The small gold represents endocytosed BSAG (5 nm). The endoplasmic reticulum (er) (A) and the Golgi complex (9) (e) are clearly labeled, whereas lamellar bodies (Ib) were negative. Bars = 0.2 pm.
acrolein, 1.25% dimethylsulfoxide in 0.1 M phosphate buffer. p H 7.4. The lungs were removed and further fixed by immersion in the same fixative. The tissue was cut into thin slices and infiltrated with increasing concentrations of gelatin up to 10% (vlv) in 0.1 M phosphate buffer. p H 7.4. at 37'C. After solidification of the gelatin at 4°C. small blocks of approximately 1 mm3 were cut and infiltrated with 2.3 M sucrose overnight at 4°C and frozen in liquid nitrogen. Ultra-thin cryosections were cut on a Reichert UltracutEIFC4E (Leica
Aktiengesellschaft; Wien. Austria) at - 100°C with a 45' cryodiamond knife (Diatome; Bienne, Switzerland) and transferred to formvar-carboncoated copper grids. Thawed ultra-thin cryosections were floated on 2% gelatin in PBS at 37°Cfor 10 min to block nonspecific binding sites, on 0.02% glycinelPBS for 10 min to block remaining free aldehyde groups. and subsequently on the primary antibody against SP-D diluted 1:50 in 1% BSAlPBS for 30-60 min. Sections were rinsed three times for 5 min with 1% BSAlPBS and immunoreactivity was visualized by incubation with
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Figure 4. lmmunogold labeling of SP-D in a rat lung Clara cell with 20-nm PAG. The lung tissue was allowed to endocytose 5-nm BSAG for 2 hr before fixation. (A) Overview of the apical part of a Clara cell with prominent labeling at the rims of secretory granules (sg). A ciliated epithelial cell (CE) is devoid of intracellular labeling. (B) Golgi complex (9) located close to the nucleus (n) which is clearly labeledfor SP-D. (C) Granule close to the nucleus (n) with a matrix density comparable to that of the cytoplasm, showing labeling for SP-D at the rim of the granule. (D) Electron-dense secretory granule near the apical plasma membrane. SP-D is clearly located at the rim of the electron dense matrix. Bars: A = 1 pm; B-D = 0.2 km.
PAC for 30 min. In double labeling experiments the last step was followed by a short rinse with PBS, fixation of the sections with 1% glutaraldehyde in PBS for 10 min, and blocking with 0,020h glycine in PBS beforc]abc]ing with the second antibody and PAG o f a different size (22). control experiments were performed by omission of the primary antibody or by using an irrelevant antibody (i.e., anti-amylase). Finally, labeled sections were rinsed four times for 5 min with distilled water, stained on a drop of uranyl acetate-oxalate, pH 7, for 5 min. and embedded in 1.8% methylcellulose containing 0.4% uranyl acetate. Sections were observed and photographed in a Jeol l2OOEX electron microscope at 80 kV.
Results I' In the rat lung Only and *macrophageswere specifically labeled with the antibody against SP-D. Alveolar Type I cells, interstitial cells, and ciliated bronchiolar epithelial cells showed no labeling for SP-D. Control experiments in which the primary antibody was omitted or substituted with an irrelevant antibody (anti-mylase)showed negligible goid labeling (data not shown). Tubular myelin, one ofthe extracellularforms of surfactant, contained no detectable SP-D (Figure 1). In alveolar
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macrophages SP-D occurred in endocytotic and lysosomal structures (Figure 2) showing endocytosed BSAG, but was absent from the ER and the Golgi complex. This suggests that intracellular SP-D derives from uptake of SP-D from the alveolar subphase. However, in both Type I1 cells and Clara cells the biosynthetic organelles were labeled (Figures 3 and 4). In Type I1 cells SP-D was found within the lumen of the ER cisternae and in the Golgi complex (Figure 3). Lamellar bodies, the intracellular storage compartment of surfactant, were almost completely devoid of labeling (Figure 3). Only 10% of the lamellar bodies (n = 113) contained a few gold particles. In addition, in sections double labeled for SP-A and SP-D both surfactant proteins co-localized in the ER and the Golgi complex of alveolar Type I1 cells, whereas lamellar bodies contained only SP-A and no SP-D (data not shown). Multivesicular bodies (MVB), which are thought to be part of the biosynthetic pathway of surfactant proteins (23). were also labeled to a limited extent, since only 20% of the MVB (n = 30) contained detectable amounts of SP-D. Although Type I1 cells were clearly labeled, the most prominent labeling was found in the Clara cells of the respiratory bronchioles (Figure 4A). Both the ER and the Golgi complex were sparsely but specifically labeled (Figure 4B). Golgi complexes were located primarily at the apical and basolateral sides of the nucleus, which in turn was situated in the basal part of the Clara cell. At the apical site of the nucleus large granules (0.2-1 pm in diameter), with a variable matrix density and surrounded by a single membrane, were profoundly labeled (Figures 4A. 4C, and 4D). The matrix density of these granules varied from a density comparable to that of the cytoplasm (Figure 4C) to very electron dense, sometimes with a crystalline appearance (Figures 4D and 8). The distribution of SP-D in the Clara cell granules was heterogeneous. SP-D labeling was found predominantly in between the granule content and the limiting membrane (Figures 4A and 4D). Neither in granules with a very dense matrix nor in granules with a loosely packed matrix was a significant amount of label found in the matrix. Most of the granules with a loosely packed matrix were found close to the nucleus, whereas granules at the upper apical part of the cell had a much more dense matrix. Occasionally exocytotic profiles of granules were encountered (Figure 5). To discriminate between the biosynthetic and the endocytotic compartments, the lung epithelial cells were allowed to endocytose BSAG continuously for 2 hr. BSAG was found in small vesicles, in MVB, and in lysosomes. The vast majority of the SP-Dcontaining granules contained no endocytosed BSAG (Figures 4-7) and did not label for the lysosomal markers LGPl20 (Figure 6) and cathepsin D (Figure 7). Occasionally a dense granule, exhibiting morphological resemblance to dense SP-D-containinggranules, contained endocytosed BSAG (Figures 6 and 7). Some of these BSAGpositive granules also contained SP-D (Figure 6). These BSAGcontaining dense granules, which occurred predominantly in the basal part of the cells, are lysosomes, since they contained LGPl20 (Figure 6) and cathepsin D (Figure 7). From previous studies it is known that the other hydrophilic surfactant protein, SP-A. is found in dense granules of rat lung Clara cells (13). In double labeling experiments it was clear that SP-A and SP-D were present in the same granules (Figures 8B8D), but within these granules SP-A and SP-D displayed different distributions.
Figure 5. Exocytotic profile of a secretory granule in a Clara cell labeled for SP-D with 20-nm PAG. Bar = 0.2 vm.
Whereas most SP-D was present at the periphery of the granule, in between the matrix and the limiting membrane, SP-A was homogeneously distributed over the matrix of the granule, indicating a spatial separation between the two surfactant proteins (Figures 8B-8D). In the biosynthetic organelles, i.e., the Golgi complex, SP-A and SP-D co-localized, whereas the labeling densities suggested that much more SP-A is present than SP-D (Figure 8A).
Discussion Pulmonary surfactant is a complex mixture of lipids and proteins (1,2). At present, four classes of proteins are considered to be specific surfactant proteins (24). These surfactant proteins are referred to as SP-A. SP-B, SP-C and SP-D according to the nomenclature proposed by Possmayer (25). The hydrophilic surfactant protein SP-A has been extensively localized at the EM level (13J7.26-28), and these studies have shown that SP-A is restricted to alveolar Type I1 cells, alveolar macrophages, tubular myelin, and Clara cells of the rat lung. In this study we have localized the structurally related SP-D using high-resolution immuno-EM. SP-D was present in alveolar Type I1 cells, alveolar macrophages, and Clara cells of the rat lung, but was absent from tubular myelin, which is in agreement with previous localization studies at the LM level (14) and the EM level (15). Although both SP-A and SP-D are synthesized and secreted by Type I1 cells, their subcellular distributions were found to differ. Whereas both proteins are present in the ER and the Golgi complex, lamellar bodies do not seem to contain SP-D in contrast to SP-A, which is present in all lamellar bodies (17). In addition, tubular myelin, one of the extracellular forms of surfactant originating from secreting lamellar bodies (4), is not labeled for SP-D. However. in a previous study we have shown that tubular myelin is heavily la-
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Figures 6 and 7. Ultra-thincryosections of Clara cells double labeled for SP-D with 15-nm PAG and the lysosomal membrane glycoprotein LGPl2O with IO-nm PAG (Figure 6) or the soluble lysosomal enzyme cathepsin D (CDA) with 10-nm PAG (Figure 7). The lung tissue was allowed to endocytose 5-nm ESAG for 2 hr before fixation. Secretory granules (sg) do not contain endocytosed ESAG and are not labeled for LGPlPO (Figure 6) or cathepsin D (Figure 7), whereas ESAG which are morphologically similar to the secretory granules, are labeled for LGPlPO (Figure 6) and cathepsin D (Figure 7) and some(arrow)-positivegranules times also contain SP-D (Figure 6). Ears = 0.5 pm.
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Figure 8. Rat lung Clara cells, fixed after 2 hr of uptake of 5 n m BSAG, double labeled for SP-D (15-nm PAG) and SP-A (10-nm PAG). (A) The Golgi complex (9) is abundantly labeled for SP-A and to a lesser extent for SP-D. (B-D) Secretory granules are homogeneously labeled for SP-A, whereas SP-D is mostly located at the rims of the granules. (D) Some granules show a crystalline matrix with the same distribution of SP-A and SP-D. Bars: A-C = 0.2 Km; D = 0.1 pm.
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beled for SP-A (17). In addition, recent studies have shown that extracellular SP-D distributes differently from SP-A. Crouch et al. (15) showed that in silica-treated rats SP-D is associated with granular material but not with tubular myelin. In alveolar lavage fluids, Kuroki et al. (29) showed that 70% of SP-D was present in the 33,000 x g supernatant, whereas about 99% of SP-A was associated with the 33,000 x g pellet, suggesting that most SP-D, although associated with lipids, is not incorporated into a large lipid-protein complex. These data, together with our localization studies, suggest that SP-D is not secreted together with the lipid-protein complex via lamellar bodies but via another secretory pathway. Since SP-A and SP-D co-localize in the Golgi complex, the diversion of SP-Dfrom the other surfactant proteins must take place somewhere between the Golgi complex and the lamellar bodies. The precise location at which SP-D is sorted from the other surfactant proteins remains to be elucidated. In the Clara cell of the rat lung, SP-D is present in the biosynthetic organelles, i.e., in the ER and the Golgi complex, and in immature and mature secretory organelles. The immature granules in the Golgi area, exhibiting a moderately dense content, seem to give rise to apically located mature secretory granules with a dense content, sometimeseven crystalline. In double labeling experiments both SP-D and SP-A were localized in the same secretory granule and are therefore secreted together via the same organelle, in contrast to the apparently different secretory routes of SP-A and SP-D in Type I1 cells. Although SP-A and SP-D were present in the same granule, they were clearly separated from each other within the same granule. The localization of SP-D at the periphery of the dense granules might be due to the exclusion of SP-D from the condensing matrix. SP-D might also have an affinity with the limiting membrane, since even in early granules SP-D is localized at the periphery of these granules. From this study and previous studies, it is evident that there are at least two secretory organelles of distinct nature that are involved in the secretion of surfactant components. Alveolar Type I1 cells have a lysosome type of secretory organelle, the lamellar body, which is connected to the endocytotic pathway. Lamellar bodies secrete the surfactant lipids together with the surfactant proteins SP-A, SP-B,and SP-C. Our immuno-EM observations suggest that lamellar bodies are devoid of SP-D. The other type of secretory organelle is found in Clara cells. These secretory organelles have no lysosomal characteristics, they are not connected to the endocytotic pathway, and they are devoid of large amounts of phospholipids, since tritiated choline is not incorporated into these granules (30). They do secrete the hydrophilic surfactant proteins SP-A and SP-D, but not the hydrophobic surfactant protein SP-B (data not shown), whereas SP-B is abundantly present in lamellar bodies (31,32). A clear function for SP-D is not known, but it seems that SP-D is not important for the structural organization of the surfactant lipids, since it is not associated with the bronchoalveolarlavage pellet (29), it is not secreted together with the surfactant lipids, it is not found in tubular myelin, and it is not able to aggregate phospholipid liposomes (29). SP-A, on the other hand, is an essential component in the structural organizationof surfactant, since it is necessary for the formation of tubular myelin in vitro (33,34), it is localized at the corners of the tubular myelin network (17), and
it is able to aggregate phospholipid liposomes (35,36). Developmental profiles of SP-D expression in rat lung (14,16) also suggest that SP-D is not needed in significant amounts before birth and that it may not be required for the formation of surface active material. In addition to its structural role in surfactant, SP-A may play an important role in the alveolar defense mechanism (21,37,38), and it might well be that SP-D, which is structurally related to SPA, conglutinin, and other C-type lectins (11,12), also has a function in the lung defense mechanism. Such a possible function of SP-D in alveolar defense has also been suggested by others (14,39,40). Since Clara cells secrete both SP-A and SP-D, and no other surfactant components, these cells could play an important role in the host-defense mechanism of the rat lung.
Acknowledgments We are grateful to Or S.Hawgood (University of Ca1;forniaSan Francisco, San Francisco)for his gift of anti SPA, to Dr K. von Figura (Georg August Universitat, Gottingen) for the anti-cathepsin D, to Dr I. Mellman pale University, New Haven) for the anti-LGPl20, and to Drs Toyoaki Akino, Hen&E! Haagsman, andJan K Slot for stimulating discussions andcritically reading the manuscript. We thank Tom van Rqn andMaurits Niekerk for preparing the micrographs.
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