Cell Tissue Res (1992) 268:277-281
Cell&Tissue Research 9 Springer-Verlag 1992
Immunolocalization of 67 kDa elastin-binding protein in perinatal rat lungs Kojiro Wasano, Yasuhiro Hirakawa, and Keiichiro Nakamura Deapartment of Anatomy, Faculty of Medicine, Kyushu University, Fukuoka, 812 Japan Received August 13, 1991 / Accepted December 28, 1991
Summary. 67 kDa elastin-binding protein (RL-67EBP) has been isolated from neonatal rat lungs by the use of an elastin-coupled affinity column, followed by elution with either lactose or synthetic elastin hexapeptide (VGVAPG), and immunohistochemistry has been used on perinatal rat lungs to determine the tissue localization of this protein. No immunoreactive structures occur in fetal lungs, or in the lungs of day-1 and -4 neonates. On day-7 after birth, immunoreactive cells appear in the subepithelial connective tissue of the intrapulmonary airways, from day-10 on, these cells become evenly distributed in the alveolar parenchyma. Occasionally, some cells occur in the alveolar air space, being free from the surface of the alveolar septum. Unpermeabilized cells obtained by bronchoalveolar lavage, show cell surface immunoreactivity, indicating that RL-67EBP is expressed on the surface membrane of the cells. From these findings, it is suggested that the immunoreactive cells are blood-borne monocytes, and that RL-67EBP may function as an elastin peptide receptor by which monocytes mobilize through interstitial connective tissue during their migration from blood to alveolar air space, where they eventually differentiate into alveolar macrophages.
differentially concentrated in specific tissue sites, and function as cell adhesion molecules by interacting with complementary glycoconjugates located at that site (Cerra et al. 1984; Leffler and Barondes 1986; Wasano et al. 1990). More recently, a new type of lectin-like galactosebinding protein, with a molecular weight of 67 kDa, has been isolated from developing bovine lungs (Barondes 1988; Hinek et at. 1988). This protein has been shown to consist of 2 distinct ligand-binding sites within a single molecule, namely, a galactose-binding and an elastinbinding site. An in vitro study (Hinek et al. 1988) of elastin-producing cells has provided evidence that these 2 ligand-binding sites play a crucial role in the macromolecular assembly of elastin subunits into mature elastic fibers. At present, however, the exact in vivo function of the lung elastin-binding protein remains unknown. In the present study, we have isolated an elastin-binding protein (RL-67EBP) from neonatal rat lungs, which resembles bovine 67 kDa elastin-binding protein in several physicochemical properties. The immunolocalization of this RL-67EBP in perinatal lung tissues has been investigated and a possible role suggested.
Materials and methods Key words: Lung - Elastin-binding protein - Lectins - Galactose - Monocytes - Immunocytochemistry - Rat (Wistar)
Endogenous soluble galactose-binding lectins have been isolated from various mammalian tissues (Barondes 1984; Drickamer 1988). Of these, the most extensively characterized are 3 rat lung lectins with distinct molecular weights of 14, 18 and 29 kDa, respectively (Cerra et al. 1985). Immunolocalization, as well as sugar-binding studies, of these lectins have suggested that they are Offprint requests to: K. Wasano
Isolation of R L - 6 7 E B P
Wistar rats of both sexes (2 weeks old) were sacrificed with intraperitoneal pentobarbital injections. The lungs were perfused with saline via the right ventricle, until free of blood. The lungs were chopped into small cubes, then homogenized in a Potter homogenizer containing l0 vol of 57 mM Na2HPO,~, 18 mM KH2PO4, 75 mM NaC1, 2 mM mercaptoethanol, 4raM EDTA, 2 mM PMSF, 5 mM benzamidine, 5 mM aminohexanoic acid (ME buffer) and 150 mM lactose, at 4~ C. After insoluble material was pelleted by centrifugation at 100000 g for 70 rain, the supernatant was exhaustively dialyzed against ME buffer without lactose, at 4~ C. The dialyzed protein solution was then applied to a 2 x 15 cm column packed with CNBr-activated Sepharose 4 B resin (Pharmacia, Sweden) coupled with elastin peptide (10 mg/ml resin) (Elastin Product, USA) and preequilibrated with ME buffer. Unbound material was removed by washing with ME buffer containing 150 mM
278 sucrose, until the protein content of the elution was negligible. After a further washing with ME buffer, bound protein was eluted by ME buffer containing either 150 mM lactose or 0.5 mg/ml synthetic elastin peptide, VGVAPG (Sigma, USA). The eluted protein was concentrated, using Amicon YM-10 ultrafiltration membranes (Grace Co., USA), to 1 ml and the amount monitored by a dyebinding method (Bradford 1976).
Isolation of 1629 kDa galactose-binding lectins 14, 18 and 29 kDa galactose-binding lectins were isolated by the same procedure as used for isolating RL-67EBP, with the exception that an asialofetuin (ASF)-immobilized affinity column was used instead of an elastin-immobilized one. The lectin with a molecular weight of 14 kDa was purified as described previously (Wasano et al. 1990) and used for control experiments.
Antibody production against RL-67EBP and rat lung 14 kDa lectin Polyclonal antisera were raised in guinea pigs. Briefly, purified RL-67EBP (10 gg per injection) was injected into the back of guinea pigs. The first booster injection was given 3 weeks later, followed by a boosters at weekly intervals. Blood was obtained from the ear veins using a 25 G needle. IgG was purified by ammonium sulfate precipitation followed by DEAE ion exchange chromatography. F(ab')2 fragments were prepared using a combination of pepsin- and protein A-immobilized columns (Pierce, USA). Anti rat lung 14 kDa lectin antibodies were raised in rabbits and characterized as described previously (Wasano et al. 1990).
Western blotting RL-67EBP or 14~29 kDa lectins isolated from lungs, were electrophoresed on polyacrylamide slab gels (Laemmli 1970) and transferred onto nitrocellulose (NC) membranes (Towbin et al. 1979). The membranes were then soaked overnight in blocking solution (BS) containing 10 mM TRIS-HCL, 500 mM NaC1, 0.05% Tween 20, 3% bovine serum albumin (BSA) and 0.1% normal goat serum (NGS), at 4~ C. Guinea pig antibodies raised against RL-67EBP or rabbit anti-rat lung 14 kDa lectin antibodies were then added. After incubation for 2 h, the membranes were washed with 3 changes (30 min each) of the same buffer without BSA and NGS, and incubated with HRP-conjugated secondary antibodies raised in goat (Cappel, USA), in BS for 2 h at room temperature. The membranes were extensively washed before color development with 4-chloro-l-naphtol/H202 solution. As a control, antibodies were replaced by the preimmune serum or adsorbed with an excessive amount of purified antigen (10 gg antigen/1 gg IgG).
Immunohistochemistry The lungs were obtained from rats at day-15 and -20 of gestation, and at 1, 4, 7, 10, 14, 21, 35 and 50 days after birth. All the lungs were irrigated with saline to remove blood, then fixed in Zamboni's fixative (Stefanini et al. 1967) containing 2% (w/v) paraformaldehyde and 15% (v/v) saturated picric acid in 0.1 M phosphate buffer (pH 7.3), for 45 rain at 4~ C. The tissues were then incubated in 15% sucrose in 0.1 M phosphate buffer (pH 7.4) and embedded into Tissue Tek (Miles, USA). Cryosections of 4 gm thickness were placed on glass slides coated with 0.1% poly-Llysine, and stored at - 8 0 ~ C. All immunohistochemical staining was performed according to Wasano et al. (1990). Sections were covered by blocking solution (BS) containing 10 mM phosphate buffer (pH 7.4), 150 mM NaCI,
1% BSA, 0.1% NGS and 1 gg/ml mouse IgG Fc fragments (Cosmo, Tokyo, Japan), for 30 rain. After removal of excess BS with tissue paper, sections were incubated for 2 h in anti-RL-67EBP IgG F(ab')2 solution diluted to 1 gg/ml with BS. The sections were then washed times with phosphate-buffered saline (PBS) and incubated for 30 rain in RITC-labeled goat anti-guinea pig IgG (Cappel, USA) diluted 100 times with BS. After washing in PBS, coverslips were mounted in glycerol gelatin and examined with an Olympus epi-illuminating fluorescence microscope BH-2 (Olympus, Japan) equipped with a dichromic mirror, B-475 excitation and G-520 emission cutting filters. After labeling with the primary anti RL67EBP antibodies, some sections were enzyme-immunolabeledusing an ABC-GO (glucose oxidase) kit (Vector, USA). The ABCGO-immunolabeled sections were dehydrated and mounted in Entellan (Merck, FRG). Some sections were double stained with a mixture of the antiRL-67EBP antibodies and anti-rat lung 14 kDa lectin antibodies. The sections were labeled with appropriate secondary antibodies conjugated with FITC and RITC, respectively. Surface labeling of cells was carried out using a broncho-alveolar lavage (BAL) technique: lungs from 2-week-old rats were irrigated with warm saline via the right ventricle to remove blood then silicone tube (Clay Adams, USA), attached to 10 ml plastic syringe containing Ca-, Mg-free Hanks' balanced salt solution (HBSS), was inserted into the trachea through a small incision made just below the larynx, and the lungs filled with HBSS. After 10 rain, the fluid was drawn back into the syringe (blood contaminated fluid was discarded) and centrifuged for 10 min at 500 g, then the cells were suspended in Dulbecco's modified Eagle medium (DMEM) and plated in 35 mm culture dishes (Corning, USA) coated with poly-L-lysine, at a density of 3 x 10r cells/dish. They were allowed to adhere for 20 min at 37~ C, in a 5% CO2 95% air atmosphere, then nonadherent cells were removed with several washing with fresh DMEM. Adherent cells were fixed with Zamboni's fixative for 5 rain on ice. After a trypan blue (molecular weight 1 kDa) exclusion test to substantiate that the cells were not permeabilized against IgG F (ab')2 fragments (molecular weight 102 kDa), the cells were stained with anti RL-67EBP IgG F(ab')2 fragments in the same manner as described above, and washed with PBS. After removal of the PBS, the dishes were examined under a fluorescence microscope by placing a coverslip onto the bottom of the culture dish. The control protocols used in Western blotting were also performed for immunohistochemistry.
Results R L - 6 7 E B P was successfully isolated f r o m the crude l u n g lactose-extract in a single passage t h r o u g h elastin-coupled affinity c o l u m n a n d e l u t i o n with lactose or V G V A P G (Fig. 1). T h e a n t i b o d i e s raised a g a i n s t the p r o t e i n stained a single 67 k D a b a n d o n the N C m e m b r a n e t r a n s b l o t t e d with the e l u t i o n (Fig. 2), i n d i c a t i n g the high specificity a n d efficiency o f the i s o l a t i o n procedure. Since b o v i n e 67 k D a e l a s t i n - b i n d i n g p r o t e i n has been s h o w n to share c o m m o n antigenic epitope(s) with l u n g 14 k D a lectin of the same species, we e x a m i n e d w h e t h e r this was also the case with R L - 6 7 E B P . W e s t e r n b l o t analysis o f p r o t e i n s (Fig. 3) eluted f r o m A S F - c o u pled affinity c o l u m n a g a i n revealed o n l y one positive b a n d at 67 k D a (Fig. 4), suggesting that the R L - 6 7 E B P was i m m u n o l o g i c a l l y distinct f r o m rat l u n g 14 k D a lectin. The immunofluorescence examinations of perinatal l u n g tissue for R L - 6 7 E B P - i m m u n o r e a c t i v e structures, showed negative results in fetal lungs a n d in the lungs o f day-1 a n d -4 n e o n a t e (Fig. 5), b u t i m m u n o r e a c t i v e
279 ficity of both the immunofluorescence and the enzymeimmunolabeling procedures employed. To further verify that RL-67EBP is immunologically distinct from rat lung 14 kDa lectin, the immunohistochemical location of both proteins was examined after using a mixture of anti RL-67EBP and anti 14 kDa rat lung lectin antibodies. As shown in Fig. 10, the 2 antibodies recognized different cell-types, thus supporting the suggestion that the RL-67EBP and rat lung 14 kDa lectin are immunologically distinct and specifically localized in their respective tissue sites. Because intense immunoreactivity was often seen at the rim o f the cells, it was presumed that RL-67EBP might be expressed on the surface membrane of the cells. This was confirmed by the surface staining of the BAL cells in the surface labeling experiment (Fig. 9).
Discussion
Fig. 1. SDS-PAGE of protein eluted from elastin affinity column with either lactose (middle lane) or synthetic elastin hexapeptide (right lane). A single protein (RL-67EBP) band slightly above the position of 66 kDa molecular weight marker (bovine albumin) (left lane) can be seen. Coomassie-blue-stained gel Fig. 2. NC paper transblotted with the elution from elastin affinity column. The paper was stained with guinea pig antisera raised against RL-67EBP. A single immunoreactive band at 67 kDa can be seen. The bar indicates the position of 66 kDa molecular weight marker (bovine albumin) Fig. 3. SDS-PAGE of proteins eluted from ASF affinity column. Three major protein bands at 14, 18 and 20 kDa can be seen. The bars indicate the positions of three molecular weight standards, lactalbumin (14 kDa), carbonic anhydrase (29 kDa) and bovine albumin (66 kDa), respectively. No apparent 67 kDa band can be observed in this picture due to the small amount of RL-67EBP compared to that of the above three proteins. Coomassie-bluestained gel Fig. 4. Immunoblot analysis of the eluted proteins from ASF affinity column with anti RL-67EBP antibodies. A single protein band at 67 kDa, which is not detectable with dye staining as shown in Fig. 3, exhibits immunoreactivity. None of the 14, 18 and 29 kDa proteins shows immunoreactivity, indicating that RL-67EBP is immunologically distinct from any of these three proteins. The bar indicates the position of bovine albumin (66 kDa)
cells had appeared by day-7 after birth and rapidly increased in number between day-7 and -14. On day-7, numerous immunoreactive cells, with a large, irregularly-contoured cell-body, were concentrated in the subepithelial connective tissue of the intrapulmonary airways (Fig. 6). From day-10 to -50, these cells were evenly scattered throughout the lung parenchyma (Fig. 7), and individual cells were often seen, free from the alveolar septurn, in the alveolar air space (Fig. 8 inset). Enzymeimmunolabeling (Fig. 8) stained cells with the same tissue distribution as the cells seen in the indirect immunofluorescence procedure. No immunoreative structure was found in any control protocol, indicating the speci-
A elastin-binding protein with a molecular weight of 67 kDa has been isolated from ligament fibroblasts as well as auricular chondroblasts of bovine origin (Hinek et al. 1988; Mecham et al. 1989), and has been shown to have an affinity not only for the elastin peptide, but also for the galactose residue. From the observation that addition of galactose into the culture medium of the elastin-producing cells significantly perturbs the appearance of mature elastic fibers in the extracellular matrix of the culture system, Hinek et al. (1988) have suggested that the two ligand-binding sites of this protein play some crucial role in the assembly of elastin precursors into mature elastic fibers. Although a similar protein has been isolated from developing bovine lungs, its exact function in vivo remains unknown. This is partly due to the lack of information concerning the cellular origin and/or behavior of the protein within the lung tissue. In the present study, neonatal rat lungs have been used as the source material for extraction of the protein, since the process of postnatal maturation of this organ has been established in detail (Burri 1974; Powell and Whitney 1980). Furthermore, due to its relatively small size, the rat lung is more easily surveyed for the diverse types of cells comprising the organ. SDS-PAGE analysis of elutants from elastin-coupled affinity column, has re, vealed a single protein band with a molecular weight of 67 kDa in neonatal rat lung tissue homogenates i which, in several physicochemical respects, is similar to the 67 kDa elastin-binding protein isolated from developing bovine lungs. The immunolocalization data demonstrate that the protein (RL-67EBP) is localized in a specific cell-type which, in shape, distribution, and postnatal development, is clearly distinguishable from other cell-types. These RL-67EBP-immunoreactive cells seem to play some important role in the postnatal development of lung tissue; they first appear and become abundant during this distinct period of lung maturation. However, it seems unlikely that the cells are involved in the elastogenesis of lung tissue, as has been suggested for bovine lung 67 k D a elastin-binding protein, for the following
280
Fig. 5. Lung tissue from day-4 neonate. No RL-67EBP immunoreactive structure can be seen. B Bronchiolar lumen, x 140
the alveolar air space free from the surface of alveolar septum. x 330
Fig. 6. Lung tissue from day-7 after birth. Strong immunoreactive cells are found in the bronchiolar subepithelial connective tissue. B Bronchiolar lumen, x 140
Fig. 9. Bronchoalveolar lavage cells from day-14 neonate lung. The cells were fixed to be unpermeabilized and stained with anti RL67EBP antibodies. Cells showing apparent surface immunoreactivity are found mingled with negative ones (arrows). x 190
Fig. 7. Lung tissue from postnatal day-14. At this stage, the immunoreactive cells are found evenly distributed in the alveolar parenchyme, x 140 Fig. 8. Lung tissue from day-35 after birth. This section was stained with enzyme-immunolabeling procedure using A B C - G O kit. Immunoreactive cells can be found in the same distribution as seen in Fig. 7. x 140. Inset: An immunoreactive cell (arrow) found in
Fig. 10A, B. A pair of fluorescence pictures from the same area of a section which was double-stained with anti RL-67EBP and anti rat lung 14 kDa lectin antibodies. The former antibodies again stain cells scattered in the alveolar parenchyma (A), whereas the latter antibodies stain smooth muscle cells surrounding airway as well as blood vessels (B). x 70
281 reasons: (1) immunoreactive cells are found in the alveolar space, and thus remote from the alveolar interstitial tissue where elastic fibers are formed; (2) no immunoreactivity occurs in any lung structure other than these cells, indicating that the protein is specifically confined to these cells and not secreted into extracellular connective tissue space; (3) the immunoreactive cells apparently persist in adult lung in which the elastogenesis has already been completed; and (4) there is no cross-immunoreactivity between RL-67EBP and rat lung 14 k D a lectin, as determined by immunoblotting and also immunohistochemical staining. This suggests that the RL-67EBP isolated in this study is not identical with the 67 k D a elastin-binding protein isolated from bovine lungs, since the latter has been shown (Hinek et al. 1988) to share a c o m m o n antigenic epitope(s) with bovine lung 14 k D a lectin. Since the present data show that RL-67EBP is expressed on the cell surface, it is possibile that the protein m a y function as a cell-surface receptor for the elastin peptide. In regard to this finding, it is of interest to note that the elastin fragment has been shown to be a potent chemoattractant for monocytes, the precursors of alveolar macrophages (Hunninghake et al. 1981). As to which site on the elastin molecule is responsible for the chemotactic activity of monocytes, evidence has been provided (Senior et al. 1984) that the chemotactic activity is associated with Val-Gly-Val-Ala-Pro-Gly ( V G V A P G ) , a repeating peptide in the tropoelastin molecule. The present results demonstrating that RL-67EBP can be eluted from an elastin-coupled column not only with lactose but also with synthetic V G V A P G peptide, indicates that it functions as an elastin receptor recognizing the V G V A P G peptide. Thus, it seems reasonable to suggest that the rapid accumulation of immunoreactive cells during postnatal lung m a t u r a t i o n represents monocytes newly mobilized into the tissue in which active elastic fiber formation takes place (Burri 1974; Powell and Whitney 1980). Monocytes m a y use the elastin receptor to move through the extracellular matrix rich in elastin or its precursors, during their migration from blood to final destination, the alveolar air space. Here they eventually differentiate in response to appropriate stimuli, into diverse functional populations of resident alveolar macrophages. However, it remains to be elucidated how the galactose-binding property of RL-67EBP affects the mechanism for cell migration through the extracellular matrix. It also remains unkown as to how monocytes, newly recruiting into adult lung tissue, use this elastin receptor for migrating through the alveolar
connective tissue in which the assembly of elastic fibers is already completed.
Acknowledgements. This work was supported by a Grant-in-Aid (no. 03670013) from the Ministry of Education, Science and Culture, Japan
References
Barondes SH (1984) Soluble lectins: a new class of extracellular proteins. Science 223 : 1259-1264 Barondes SH (1988) Bifunctional properties of lectins : lectins redefined. Trends Biochem Sci 13:480-482 Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein using the principle of protein-dye binding. Anal Biochem 72: 248-254 Burri PH (1974) The postnatal growth of the rat lung. Ill Morphology. Anat Rec 180:77-96 Cerra RF, Haywood-Reid PL, Barondes SH (1984) Endogenous mammalian lectin localized extracellularly in lung elastic fibers. J Cell Biol 98:1580-1589 Cerra RF, Gitt MA, Barondes SH (1985) Three soluble rat pgalactoside-binding lectins. J Biol Chem 260 : 10474-10477 Drickamer K (1988) Two distinct classes of carbohydrate-recognb tion domains in animal lectins. J Biol Chem 263:955%9560 Hinek A, Wrenn DS, Mecham RP, Barondes SH (1988) The elastin receptor: a galactoside-binding protein. Science 249 : 1539-1541 Hunninghake GW, Davidson JM, Rennard S, Szapiel S, Gadek JE, Crystal RG (1981) Elastin fragments attract macrophage precursors to diseased sites in pulmonary emphysema. Science 212 :925-927 Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriphage T4. Nature 227: 680-685 Leffler H, Barondes SH (1986) Specificity of binding of three rat lung lectins to substituted and unsubstituted mammalian /~galactoside. J Biol Chem 261:10119-10126 Mecham RP, Hinek A, Entwistle R, Wrenn DS, Griffin GL, Senior RM (1989) Elastin binds to multifunctional 67-kilodalton peripheral membrane protein. Biochemistry 28:3716-3722 Powell JT, Whitney PL (1980) Postnatal development of rat lung: changes in lung lectin, elastin, acetylcholinesterase and other enzymes. Biochem J 188 : 1 8 Senior RM, Griffin GL, Mecham RP, Wrenn DS, Prasad KU, Urry DW (I984) Val-Gly-Val-Ala-Pro-Gly, a repeating peptide in elastin, is chemotactic for fibroblasts and monocytes. J Cell Biol 99 : 870-874 Stefanini M, De Martio C, Zamboni C (1967) Fixation of ejaculated spermatozoa for electron microscopy. Nature 216:173 174 Towbin H, Stehelin T, Gordon J (1979) Electrophoretie transfer of proteins from polyaerylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76:4350-4354 Wasano K, Hirakwa Y, Yamamoto T (1990) Immunolocalization of 14 kDa fl-galactoside-binding lectin in various organs of rat, Cell Tissue Res 259:43-49
Cell&Tissue Research 9 Springer-Verlag 1992
Immunolocalization of 67 kDa elastin-binding protein in perinatal rat lungs Kojiro Wasano, Yasuhiro Hirakawa, and Keiichiro Nakamura Deapartment of Anatomy, Faculty of Medicine, Kyushu University, Fukuoka, 812 Japan Received August 13, 1991 / Accepted December 28, 1991
Summary. 67 kDa elastin-binding protein (RL-67EBP) has been isolated from neonatal rat lungs by the use of an elastin-coupled affinity column, followed by elution with either lactose or synthetic elastin hexapeptide (VGVAPG), and immunohistochemistry has been used on perinatal rat lungs to determine the tissue localization of this protein. No immunoreactive structures occur in fetal lungs, or in the lungs of day-1 and -4 neonates. On day-7 after birth, immunoreactive cells appear in the subepithelial connective tissue of the intrapulmonary airways, from day-10 on, these cells become evenly distributed in the alveolar parenchyma. Occasionally, some cells occur in the alveolar air space, being free from the surface of the alveolar septum. Unpermeabilized cells obtained by bronchoalveolar lavage, show cell surface immunoreactivity, indicating that RL-67EBP is expressed on the surface membrane of the cells. From these findings, it is suggested that the immunoreactive cells are blood-borne monocytes, and that RL-67EBP may function as an elastin peptide receptor by which monocytes mobilize through interstitial connective tissue during their migration from blood to alveolar air space, where they eventually differentiate into alveolar macrophages.
differentially concentrated in specific tissue sites, and function as cell adhesion molecules by interacting with complementary glycoconjugates located at that site (Cerra et al. 1984; Leffler and Barondes 1986; Wasano et al. 1990). More recently, a new type of lectin-like galactosebinding protein, with a molecular weight of 67 kDa, has been isolated from developing bovine lungs (Barondes 1988; Hinek et at. 1988). This protein has been shown to consist of 2 distinct ligand-binding sites within a single molecule, namely, a galactose-binding and an elastinbinding site. An in vitro study (Hinek et al. 1988) of elastin-producing cells has provided evidence that these 2 ligand-binding sites play a crucial role in the macromolecular assembly of elastin subunits into mature elastic fibers. At present, however, the exact in vivo function of the lung elastin-binding protein remains unknown. In the present study, we have isolated an elastin-binding protein (RL-67EBP) from neonatal rat lungs, which resembles bovine 67 kDa elastin-binding protein in several physicochemical properties. The immunolocalization of this RL-67EBP in perinatal lung tissues has been investigated and a possible role suggested.
Materials and methods Key words: Lung - Elastin-binding protein - Lectins - Galactose - Monocytes - Immunocytochemistry - Rat (Wistar)
Endogenous soluble galactose-binding lectins have been isolated from various mammalian tissues (Barondes 1984; Drickamer 1988). Of these, the most extensively characterized are 3 rat lung lectins with distinct molecular weights of 14, 18 and 29 kDa, respectively (Cerra et al. 1985). Immunolocalization, as well as sugar-binding studies, of these lectins have suggested that they are Offprint requests to: K. Wasano
Isolation of R L - 6 7 E B P
Wistar rats of both sexes (2 weeks old) were sacrificed with intraperitoneal pentobarbital injections. The lungs were perfused with saline via the right ventricle, until free of blood. The lungs were chopped into small cubes, then homogenized in a Potter homogenizer containing l0 vol of 57 mM Na2HPO,~, 18 mM KH2PO4, 75 mM NaC1, 2 mM mercaptoethanol, 4raM EDTA, 2 mM PMSF, 5 mM benzamidine, 5 mM aminohexanoic acid (ME buffer) and 150 mM lactose, at 4~ C. After insoluble material was pelleted by centrifugation at 100000 g for 70 rain, the supernatant was exhaustively dialyzed against ME buffer without lactose, at 4~ C. The dialyzed protein solution was then applied to a 2 x 15 cm column packed with CNBr-activated Sepharose 4 B resin (Pharmacia, Sweden) coupled with elastin peptide (10 mg/ml resin) (Elastin Product, USA) and preequilibrated with ME buffer. Unbound material was removed by washing with ME buffer containing 150 mM
278 sucrose, until the protein content of the elution was negligible. After a further washing with ME buffer, bound protein was eluted by ME buffer containing either 150 mM lactose or 0.5 mg/ml synthetic elastin peptide, VGVAPG (Sigma, USA). The eluted protein was concentrated, using Amicon YM-10 ultrafiltration membranes (Grace Co., USA), to 1 ml and the amount monitored by a dyebinding method (Bradford 1976).
Isolation of 1629 kDa galactose-binding lectins 14, 18 and 29 kDa galactose-binding lectins were isolated by the same procedure as used for isolating RL-67EBP, with the exception that an asialofetuin (ASF)-immobilized affinity column was used instead of an elastin-immobilized one. The lectin with a molecular weight of 14 kDa was purified as described previously (Wasano et al. 1990) and used for control experiments.
Antibody production against RL-67EBP and rat lung 14 kDa lectin Polyclonal antisera were raised in guinea pigs. Briefly, purified RL-67EBP (10 gg per injection) was injected into the back of guinea pigs. The first booster injection was given 3 weeks later, followed by a boosters at weekly intervals. Blood was obtained from the ear veins using a 25 G needle. IgG was purified by ammonium sulfate precipitation followed by DEAE ion exchange chromatography. F(ab')2 fragments were prepared using a combination of pepsin- and protein A-immobilized columns (Pierce, USA). Anti rat lung 14 kDa lectin antibodies were raised in rabbits and characterized as described previously (Wasano et al. 1990).
Western blotting RL-67EBP or 14~29 kDa lectins isolated from lungs, were electrophoresed on polyacrylamide slab gels (Laemmli 1970) and transferred onto nitrocellulose (NC) membranes (Towbin et al. 1979). The membranes were then soaked overnight in blocking solution (BS) containing 10 mM TRIS-HCL, 500 mM NaC1, 0.05% Tween 20, 3% bovine serum albumin (BSA) and 0.1% normal goat serum (NGS), at 4~ C. Guinea pig antibodies raised against RL-67EBP or rabbit anti-rat lung 14 kDa lectin antibodies were then added. After incubation for 2 h, the membranes were washed with 3 changes (30 min each) of the same buffer without BSA and NGS, and incubated with HRP-conjugated secondary antibodies raised in goat (Cappel, USA), in BS for 2 h at room temperature. The membranes were extensively washed before color development with 4-chloro-l-naphtol/H202 solution. As a control, antibodies were replaced by the preimmune serum or adsorbed with an excessive amount of purified antigen (10 gg antigen/1 gg IgG).
Immunohistochemistry The lungs were obtained from rats at day-15 and -20 of gestation, and at 1, 4, 7, 10, 14, 21, 35 and 50 days after birth. All the lungs were irrigated with saline to remove blood, then fixed in Zamboni's fixative (Stefanini et al. 1967) containing 2% (w/v) paraformaldehyde and 15% (v/v) saturated picric acid in 0.1 M phosphate buffer (pH 7.3), for 45 rain at 4~ C. The tissues were then incubated in 15% sucrose in 0.1 M phosphate buffer (pH 7.4) and embedded into Tissue Tek (Miles, USA). Cryosections of 4 gm thickness were placed on glass slides coated with 0.1% poly-Llysine, and stored at - 8 0 ~ C. All immunohistochemical staining was performed according to Wasano et al. (1990). Sections were covered by blocking solution (BS) containing 10 mM phosphate buffer (pH 7.4), 150 mM NaCI,
1% BSA, 0.1% NGS and 1 gg/ml mouse IgG Fc fragments (Cosmo, Tokyo, Japan), for 30 rain. After removal of excess BS with tissue paper, sections were incubated for 2 h in anti-RL-67EBP IgG F(ab')2 solution diluted to 1 gg/ml with BS. The sections were then washed times with phosphate-buffered saline (PBS) and incubated for 30 rain in RITC-labeled goat anti-guinea pig IgG (Cappel, USA) diluted 100 times with BS. After washing in PBS, coverslips were mounted in glycerol gelatin and examined with an Olympus epi-illuminating fluorescence microscope BH-2 (Olympus, Japan) equipped with a dichromic mirror, B-475 excitation and G-520 emission cutting filters. After labeling with the primary anti RL67EBP antibodies, some sections were enzyme-immunolabeledusing an ABC-GO (glucose oxidase) kit (Vector, USA). The ABCGO-immunolabeled sections were dehydrated and mounted in Entellan (Merck, FRG). Some sections were double stained with a mixture of the antiRL-67EBP antibodies and anti-rat lung 14 kDa lectin antibodies. The sections were labeled with appropriate secondary antibodies conjugated with FITC and RITC, respectively. Surface labeling of cells was carried out using a broncho-alveolar lavage (BAL) technique: lungs from 2-week-old rats were irrigated with warm saline via the right ventricle to remove blood then silicone tube (Clay Adams, USA), attached to 10 ml plastic syringe containing Ca-, Mg-free Hanks' balanced salt solution (HBSS), was inserted into the trachea through a small incision made just below the larynx, and the lungs filled with HBSS. After 10 rain, the fluid was drawn back into the syringe (blood contaminated fluid was discarded) and centrifuged for 10 min at 500 g, then the cells were suspended in Dulbecco's modified Eagle medium (DMEM) and plated in 35 mm culture dishes (Corning, USA) coated with poly-L-lysine, at a density of 3 x 10r cells/dish. They were allowed to adhere for 20 min at 37~ C, in a 5% CO2 95% air atmosphere, then nonadherent cells were removed with several washing with fresh DMEM. Adherent cells were fixed with Zamboni's fixative for 5 rain on ice. After a trypan blue (molecular weight 1 kDa) exclusion test to substantiate that the cells were not permeabilized against IgG F (ab')2 fragments (molecular weight 102 kDa), the cells were stained with anti RL-67EBP IgG F(ab')2 fragments in the same manner as described above, and washed with PBS. After removal of the PBS, the dishes were examined under a fluorescence microscope by placing a coverslip onto the bottom of the culture dish. The control protocols used in Western blotting were also performed for immunohistochemistry.
Results R L - 6 7 E B P was successfully isolated f r o m the crude l u n g lactose-extract in a single passage t h r o u g h elastin-coupled affinity c o l u m n a n d e l u t i o n with lactose or V G V A P G (Fig. 1). T h e a n t i b o d i e s raised a g a i n s t the p r o t e i n stained a single 67 k D a b a n d o n the N C m e m b r a n e t r a n s b l o t t e d with the e l u t i o n (Fig. 2), i n d i c a t i n g the high specificity a n d efficiency o f the i s o l a t i o n procedure. Since b o v i n e 67 k D a e l a s t i n - b i n d i n g p r o t e i n has been s h o w n to share c o m m o n antigenic epitope(s) with l u n g 14 k D a lectin of the same species, we e x a m i n e d w h e t h e r this was also the case with R L - 6 7 E B P . W e s t e r n b l o t analysis o f p r o t e i n s (Fig. 3) eluted f r o m A S F - c o u pled affinity c o l u m n a g a i n revealed o n l y one positive b a n d at 67 k D a (Fig. 4), suggesting that the R L - 6 7 E B P was i m m u n o l o g i c a l l y distinct f r o m rat l u n g 14 k D a lectin. The immunofluorescence examinations of perinatal l u n g tissue for R L - 6 7 E B P - i m m u n o r e a c t i v e structures, showed negative results in fetal lungs a n d in the lungs o f day-1 a n d -4 n e o n a t e (Fig. 5), b u t i m m u n o r e a c t i v e
279 ficity of both the immunofluorescence and the enzymeimmunolabeling procedures employed. To further verify that RL-67EBP is immunologically distinct from rat lung 14 kDa lectin, the immunohistochemical location of both proteins was examined after using a mixture of anti RL-67EBP and anti 14 kDa rat lung lectin antibodies. As shown in Fig. 10, the 2 antibodies recognized different cell-types, thus supporting the suggestion that the RL-67EBP and rat lung 14 kDa lectin are immunologically distinct and specifically localized in their respective tissue sites. Because intense immunoreactivity was often seen at the rim o f the cells, it was presumed that RL-67EBP might be expressed on the surface membrane of the cells. This was confirmed by the surface staining of the BAL cells in the surface labeling experiment (Fig. 9).
Discussion
Fig. 1. SDS-PAGE of protein eluted from elastin affinity column with either lactose (middle lane) or synthetic elastin hexapeptide (right lane). A single protein (RL-67EBP) band slightly above the position of 66 kDa molecular weight marker (bovine albumin) (left lane) can be seen. Coomassie-blue-stained gel Fig. 2. NC paper transblotted with the elution from elastin affinity column. The paper was stained with guinea pig antisera raised against RL-67EBP. A single immunoreactive band at 67 kDa can be seen. The bar indicates the position of 66 kDa molecular weight marker (bovine albumin) Fig. 3. SDS-PAGE of proteins eluted from ASF affinity column. Three major protein bands at 14, 18 and 20 kDa can be seen. The bars indicate the positions of three molecular weight standards, lactalbumin (14 kDa), carbonic anhydrase (29 kDa) and bovine albumin (66 kDa), respectively. No apparent 67 kDa band can be observed in this picture due to the small amount of RL-67EBP compared to that of the above three proteins. Coomassie-bluestained gel Fig. 4. Immunoblot analysis of the eluted proteins from ASF affinity column with anti RL-67EBP antibodies. A single protein band at 67 kDa, which is not detectable with dye staining as shown in Fig. 3, exhibits immunoreactivity. None of the 14, 18 and 29 kDa proteins shows immunoreactivity, indicating that RL-67EBP is immunologically distinct from any of these three proteins. The bar indicates the position of bovine albumin (66 kDa)
cells had appeared by day-7 after birth and rapidly increased in number between day-7 and -14. On day-7, numerous immunoreactive cells, with a large, irregularly-contoured cell-body, were concentrated in the subepithelial connective tissue of the intrapulmonary airways (Fig. 6). From day-10 to -50, these cells were evenly scattered throughout the lung parenchyma (Fig. 7), and individual cells were often seen, free from the alveolar septurn, in the alveolar air space (Fig. 8 inset). Enzymeimmunolabeling (Fig. 8) stained cells with the same tissue distribution as the cells seen in the indirect immunofluorescence procedure. No immunoreative structure was found in any control protocol, indicating the speci-
A elastin-binding protein with a molecular weight of 67 kDa has been isolated from ligament fibroblasts as well as auricular chondroblasts of bovine origin (Hinek et al. 1988; Mecham et al. 1989), and has been shown to have an affinity not only for the elastin peptide, but also for the galactose residue. From the observation that addition of galactose into the culture medium of the elastin-producing cells significantly perturbs the appearance of mature elastic fibers in the extracellular matrix of the culture system, Hinek et al. (1988) have suggested that the two ligand-binding sites of this protein play some crucial role in the assembly of elastin precursors into mature elastic fibers. Although a similar protein has been isolated from developing bovine lungs, its exact function in vivo remains unknown. This is partly due to the lack of information concerning the cellular origin and/or behavior of the protein within the lung tissue. In the present study, neonatal rat lungs have been used as the source material for extraction of the protein, since the process of postnatal maturation of this organ has been established in detail (Burri 1974; Powell and Whitney 1980). Furthermore, due to its relatively small size, the rat lung is more easily surveyed for the diverse types of cells comprising the organ. SDS-PAGE analysis of elutants from elastin-coupled affinity column, has re, vealed a single protein band with a molecular weight of 67 kDa in neonatal rat lung tissue homogenates i which, in several physicochemical respects, is similar to the 67 kDa elastin-binding protein isolated from developing bovine lungs. The immunolocalization data demonstrate that the protein (RL-67EBP) is localized in a specific cell-type which, in shape, distribution, and postnatal development, is clearly distinguishable from other cell-types. These RL-67EBP-immunoreactive cells seem to play some important role in the postnatal development of lung tissue; they first appear and become abundant during this distinct period of lung maturation. However, it seems unlikely that the cells are involved in the elastogenesis of lung tissue, as has been suggested for bovine lung 67 k D a elastin-binding protein, for the following
280
Fig. 5. Lung tissue from day-4 neonate. No RL-67EBP immunoreactive structure can be seen. B Bronchiolar lumen, x 140
the alveolar air space free from the surface of alveolar septum. x 330
Fig. 6. Lung tissue from day-7 after birth. Strong immunoreactive cells are found in the bronchiolar subepithelial connective tissue. B Bronchiolar lumen, x 140
Fig. 9. Bronchoalveolar lavage cells from day-14 neonate lung. The cells were fixed to be unpermeabilized and stained with anti RL67EBP antibodies. Cells showing apparent surface immunoreactivity are found mingled with negative ones (arrows). x 190
Fig. 7. Lung tissue from postnatal day-14. At this stage, the immunoreactive cells are found evenly distributed in the alveolar parenchyme, x 140 Fig. 8. Lung tissue from day-35 after birth. This section was stained with enzyme-immunolabeling procedure using A B C - G O kit. Immunoreactive cells can be found in the same distribution as seen in Fig. 7. x 140. Inset: An immunoreactive cell (arrow) found in
Fig. 10A, B. A pair of fluorescence pictures from the same area of a section which was double-stained with anti RL-67EBP and anti rat lung 14 kDa lectin antibodies. The former antibodies again stain cells scattered in the alveolar parenchyma (A), whereas the latter antibodies stain smooth muscle cells surrounding airway as well as blood vessels (B). x 70
281 reasons: (1) immunoreactive cells are found in the alveolar space, and thus remote from the alveolar interstitial tissue where elastic fibers are formed; (2) no immunoreactivity occurs in any lung structure other than these cells, indicating that the protein is specifically confined to these cells and not secreted into extracellular connective tissue space; (3) the immunoreactive cells apparently persist in adult lung in which the elastogenesis has already been completed; and (4) there is no cross-immunoreactivity between RL-67EBP and rat lung 14 k D a lectin, as determined by immunoblotting and also immunohistochemical staining. This suggests that the RL-67EBP isolated in this study is not identical with the 67 k D a elastin-binding protein isolated from bovine lungs, since the latter has been shown (Hinek et al. 1988) to share a c o m m o n antigenic epitope(s) with bovine lung 14 k D a lectin. Since the present data show that RL-67EBP is expressed on the cell surface, it is possibile that the protein m a y function as a cell-surface receptor for the elastin peptide. In regard to this finding, it is of interest to note that the elastin fragment has been shown to be a potent chemoattractant for monocytes, the precursors of alveolar macrophages (Hunninghake et al. 1981). As to which site on the elastin molecule is responsible for the chemotactic activity of monocytes, evidence has been provided (Senior et al. 1984) that the chemotactic activity is associated with Val-Gly-Val-Ala-Pro-Gly ( V G V A P G ) , a repeating peptide in the tropoelastin molecule. The present results demonstrating that RL-67EBP can be eluted from an elastin-coupled column not only with lactose but also with synthetic V G V A P G peptide, indicates that it functions as an elastin receptor recognizing the V G V A P G peptide. Thus, it seems reasonable to suggest that the rapid accumulation of immunoreactive cells during postnatal lung m a t u r a t i o n represents monocytes newly mobilized into the tissue in which active elastic fiber formation takes place (Burri 1974; Powell and Whitney 1980). Monocytes m a y use the elastin receptor to move through the extracellular matrix rich in elastin or its precursors, during their migration from blood to final destination, the alveolar air space. Here they eventually differentiate in response to appropriate stimuli, into diverse functional populations of resident alveolar macrophages. However, it remains to be elucidated how the galactose-binding property of RL-67EBP affects the mechanism for cell migration through the extracellular matrix. It also remains unkown as to how monocytes, newly recruiting into adult lung tissue, use this elastin receptor for migrating through the alveolar
connective tissue in which the assembly of elastic fibers is already completed.
Acknowledgements. This work was supported by a Grant-in-Aid (no. 03670013) from the Ministry of Education, Science and Culture, Japan
References
Barondes SH (1984) Soluble lectins: a new class of extracellular proteins. Science 223 : 1259-1264 Barondes SH (1988) Bifunctional properties of lectins : lectins redefined. Trends Biochem Sci 13:480-482 Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein using the principle of protein-dye binding. Anal Biochem 72: 248-254 Burri PH (1974) The postnatal growth of the rat lung. Ill Morphology. Anat Rec 180:77-96 Cerra RF, Haywood-Reid PL, Barondes SH (1984) Endogenous mammalian lectin localized extracellularly in lung elastic fibers. J Cell Biol 98:1580-1589 Cerra RF, Gitt MA, Barondes SH (1985) Three soluble rat pgalactoside-binding lectins. J Biol Chem 260 : 10474-10477 Drickamer K (1988) Two distinct classes of carbohydrate-recognb tion domains in animal lectins. J Biol Chem 263:955%9560 Hinek A, Wrenn DS, Mecham RP, Barondes SH (1988) The elastin receptor: a galactoside-binding protein. Science 249 : 1539-1541 Hunninghake GW, Davidson JM, Rennard S, Szapiel S, Gadek JE, Crystal RG (1981) Elastin fragments attract macrophage precursors to diseased sites in pulmonary emphysema. Science 212 :925-927 Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriphage T4. Nature 227: 680-685 Leffler H, Barondes SH (1986) Specificity of binding of three rat lung lectins to substituted and unsubstituted mammalian /~galactoside. J Biol Chem 261:10119-10126 Mecham RP, Hinek A, Entwistle R, Wrenn DS, Griffin GL, Senior RM (1989) Elastin binds to multifunctional 67-kilodalton peripheral membrane protein. Biochemistry 28:3716-3722 Powell JT, Whitney PL (1980) Postnatal development of rat lung: changes in lung lectin, elastin, acetylcholinesterase and other enzymes. Biochem J 188 : 1 8 Senior RM, Griffin GL, Mecham RP, Wrenn DS, Prasad KU, Urry DW (I984) Val-Gly-Val-Ala-Pro-Gly, a repeating peptide in elastin, is chemotactic for fibroblasts and monocytes. J Cell Biol 99 : 870-874 Stefanini M, De Martio C, Zamboni C (1967) Fixation of ejaculated spermatozoa for electron microscopy. Nature 216:173 174 Towbin H, Stehelin T, Gordon J (1979) Electrophoretie transfer of proteins from polyaerylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76:4350-4354 Wasano K, Hirakwa Y, Yamamoto T (1990) Immunolocalization of 14 kDa fl-galactoside-binding lectin in various organs of rat, Cell Tissue Res 259:43-49
