Morphogenetic and functional activity of type II cells in early fetal rhesus monkey lungs.код для вставкиСкачать
THE ANATOMICAL RECORD 234:93-104 (1992) Morphoge ietic and Functional Activity of Type II Cells in Early Fetal Rhesus Monkey Lungs A COMPARISON BETWEEN PRIMATES AND RODENTS ANK A.W. TEN HAVE-OPBROEK AND CHARLES G. PLOPPER Department of Pulmonology, School of Medicine, University of Leiden, P.O. Box 9602,2300 RC Leiden, The Netherlands (A.A.W.T.H.-0.);Department of Anatomy, School of Veterinary Medicine, University of California, Davis, California 95616 (C.G.P.) ABSTRACT To evaluate further the role of type I1 alveolar epithelial cells in primate lung development, lungs of fetal (46 to 155 days gestational age [DGA]), postnatal, and adult rhesus monkeys were investigated with antibodies against surfactant protein A (SP-A), Alcian blue (AB) staining, and periodic acid-Schiff (PAS) staining with/without alpha-amylase pre-treatment. In adult and postnatal lungs, type I1 cells (cuboid shape; large, roundish nucleus) displayed a unique cytoplasmic staining for SP-A. In prenatal lungs, a low-columnar to cuboid type of cell with a large, roundish nucleus was first detectable by 62 DGA. It was the only cell type to line the distalmost tubules or buds of the prospective respiratory tract. It exhibited (initially partial) cytoplasmic staining for SP-A. AB and PAS stainings showed the presence of acid glycoconjugates and large apical and/or basal glycogen fields. After 95 DGA, the lining of the distal respiratory tract additionally displayed flatter cells with immunoreactivity for SP-A and non-reactive zones. Columnar epithelium (pseudostratified or simple) never stained for SP-A. We conclude that morphologically identifiable type I1 cells first appear in fetal rhesus monkey lungs by 62 DGA (pseudoglandular period). The cells may already synthesize surfactant and extracellular matrix components. They generate type I cells, and thus the entire pulmonary acinus lining. These conclusions for the rhesus monkey fully agree with our earlier conclusions for another primate, the human, and for rodents. However, as presently shown, primates differ greatly from rodents with respect to the timing of type I1 cell differentiation (at 29-38% versus 73-75% of gestation or at 22-25% versus 48-49% of prenatal lung development). 0 1992 Wiley-Liss, Inc. It has been thought for a long time (reviews by Ten I1 cell differentiation and function (i.e., surfactant synHave-Opbroek, 1975, 1979) that type I1 alveolar epi- thesis) start at a much earlier stage of lung developthelial cells first appear prenatally at about the same ment. These studies first detect type I1 cells in embryos time as type I alveolar epithelial cells, that is, in the with a developmental age (basis: embryonic weight, period just before birth when primitive alveoli (smooth- Ten Have-Opbroek, 1975) of 14.2 days, i.e., as early as walled ducts and sacs) are formed (terminal-sac pe- the pseudoglandular period. The early type I1 cells disriod). At that time, cuboid cells lining these ducts and play distinctive features, i.e., a low-columnar to cuboid sacs start to display multilamellar bodies (MLBI-tracell shape, a large and roundish nucleus, a unique cyditional markers of type I1 cells. MLB are organelles of toplasmic staining for surfactant-associated protein A, the endocytic pathway that lead to secretion of pulmo- SP-A, and postulated precursory stages of MLB, includnary surfactant, a complex lipid-protein substance that ing a potentially primary structure in prenatal MLB reduces surface tension at the airlliquid interface and formation (Ten Have-Opbroek et al., 1988, 1990b), i.e., thereby, provides mechanical stability to the pulmo- a cytoplasmic inclusion filled with glycogen. Mouse nary acini (review by Ten Have-Opbroek et al., 1990b). Clara cells display different developmental and funcAlso, based on the appearance of MLB, type I1 cells tional patterns (Ten Have-Opbroek, 1991; Ten Havehave been assumed to start with surfactant synthesis Opbroek and DeVries, 1991). First, they acquire their in the terminal-sac period (review by Ten Have-Op- adult phenotype and SP-A reactivity only after birth. Second, their staining pattern for SP-A (apical cap) broek, 1975). Our light microscopic (Ten Have-Opbroek, 1975, 1979, 1981, 1991; Van Hemert et al., 1986; Oomen et al., 1990) and electron microscopic (Ten Have-Opbroek et al., 1988,1990b; Ten Have-Opbroek, 1991) studies in Received August 28, 1991; accepted January 15, 1992. the mouse embryo have been the first to show that type ~~ 0 1992 WILEY-LISS. INC. 94 A.A.W. TEN HAVE-OPBROEK AND C.G. PLOPPER suggests internalization of SP-A from distal air spaces during postnatal and adult life. As proposed and discussed (Ten Have-Opbroek et al., 1988,1991),we use the general term type I1 cell to indicate this cell type pre- and postnatally. Our hypothesis based on studies in the mouse-i.e., that onset of type I1 cell differentiation and function in mammals takes place in the pseudoglandular period of lung development (Ten Have-Opbroek, 1981)-is fully supported by our studies in another rodent, the rat (Otto-Verberne and Ten Have-Opbroek, 1987),and in humans (Otto-Verberne et al., 1988, 1990).Other investigators also report on type I1 cell differentiation in rodents, based on studies with biochemical cell markers (Jaskoll et al., 1984,1988;Funkhouser et al., 1987; Edelson et al., 1988;Paine et al., 1988;Slavkin et al., 1989; Joyce-Brady and Brody, 1990; Williams and Dobbs, 1990;Woodcock-Mitchell et al., 19901,or ultrastructural, morphometric techniques (Lin and Lechner, 1990;Snyder and Magliato, 1991;Young et al., 1991). However, as will be discussed, a close comparison with our results was often not possible. The biochemical results were frequently not related to specific epithelial cell types in developing lungs. Some studies started at late gestational ages. The present study was performed to find out if the early onset of type I1 cell differentiation and function is also true for another primate, the rhesus monkey. We examined fetal, postnatal, and adult rhesus monkey lungs with light microscopic immunocytochemistry, using our polyclonal antibodies against human or mouse surfactant protein, and carbohydrate cytochemistry, using periodic acid Schiff (PAS), alpha-amylase/ PAS, and Alcian blue (AB). Our present study indicates that morphologically identifiable and functionally active type I1 cells first appear in rhesus monkeys by 62 DGA (pseudoglandular period of lung development). These cells play a primary role in pulmonary acinus development, and give rise to type I cells. In general, rhesus monkey lung development follows the pathway proposed for mammals (Ten Have-Opbroek, 1981, 1991).However, primates differ from rodents with respect to the timing of type I1 cell differentiation (at 29-38% versus 73-75% of the gestation time or at 22-25% versus 48-49% of the total time of prenatal lung development). Some of the observations have been published in an abstract (Ten Have-Opbroek and Plopper, 1989). MATERIALS AND METHODS Lung Material, Fixation Procedures Lung material from fetal, postnatal, and adult rhesus monkeys (Macaca mulatta, gestation time ca. 165 days) was obtained as described (Tyler et al., 1988; Plopper et al., 19891,and selected from a collection of 51 fetal (29-165 days gestational age, DGA), eight postnatal, and five adult monkey lungs. For immunofluorescence, use was made of 11 fetuses aged between 46 and 142 DGA; three animals aged 6, 8,and 9 days postnatally (dPN); and three adult animals. Immediately after removal of the lungs from the thorax, tissue blocks were cut from the peripheral parenchyma and briefly rinsed in PBS (pH 7.2).Some of these blocks were prepared and used for cryostat sectioning. The fresh-frozen sections (ca. 6 km) were usu- ally fixed in pro-analysis acetone (Merck, Germany) at -20°C for 5-10 min (Otto-Verberne and Ten Have-Opbroek, 19871,but sometimes they were only heated at 60°C for 3-5 min to preserve the surfactant lining layer along the air spaces. Other tissue blocks were fixed by immersion in Clark's acid (ethanol 100% and acetic acid, 3:l v/v) or in 1% paraformaldehyde or 1% paraformaldehyde and 0.1% glutaraldehyde in cacodylate buffer (pH 7.2).After dehydration and embedding in paraffin, the fixed blocks were sectioned at 6 bm thickness. The same lung material was used for the PAP technique and carbohydrate cytochemistry. In addition, use was made of paraffin-embedded lung sections derived from 73 to 155 DGA-old-fetusesand fixed via tracheal infusion of a mixture of paraformaldehyde and glutaraldehyde in cacodylate buffer (350-400 mOsm, pH 7.2) (Karnovsky, 1965) at 30 cm fixative pressure as reported (Tyler et al., 1988;Plopper et al., 1989). Sections stained with hematoxylin and eosin (H&E) were used to select blocks for (immuno)cytochemistry as well as for general orientation. Antibodies Specific antibodies recognizing type I1 cells on the basis of a unique, cytoplasmic staining were prepared in our laboratory by injecting rabbits with the pellet fraction of bronchoalveolar lavage fluid from adult human lung (Otto-Verberne et al., 1988).The resulting immune serum was absorbed with serum and crossreacting organs (Otto-Verberne et al., 1988).The SP-A specificity of the final immune serum, called specific anti-lavage serum, Human (SALS-Hu)was assessed by Western blot analysis, in vitro translation assays, and immunohistochemistry of relevant material (serum, tissues, organs; fetal/adult) from humans, and also from some other species including the dog (criteriaSP-A: molecular mass ca. 35 kD; selective, cytoplasmic staining of type I1 cells; selective blocking of SP-A immunoreactivity by pre-absorption of SALS-Hu with recombinant SP-A, see Otto-Verberne et al., 1988,1990, 1991;Ten Have-Opbroek et al., 1990a, 1991).In addition, we used one of our specific anti-mouse reagents, SAALS (specific anti-adult lung serum, rabbit antimouse) as a positive control. SAALS specifically reacts to SP-A and a probably surfactant-related 12 kD protein, and marks type I1 cells in several species strongly by a unique, cytoplasmic staining, as shown in mice (Ten Have-Opbroek, 1975,1979,1981;Van Hemert et al., 19861,rats (Otto-Verberne and Ten Have-Opbroek, 1987; Batenburg et al., 1988), and humans (unpublished observations). /mmunoc~ocbemis~~ lmmunofluorescence Lung sections were incubated with SALS-Hu or SAALS for 60 min, and then for 45 min with fluorescein isothiocyanate-conjugated swine anti-rabbit IgG (150 in PBS, pH 7.2) (Dakopatts, Glostrup, DK; absorbed with human IgG), both incubations being performed at room temperature (RT). Before and after each incubation the sections were washed in 3-4 changes of PBS (pH 7.2)for 15-20 min. The sections were mounted under a coverslip in a mixture of 80% glycerol and 20% PBS (pH 8.0) containing p-phenylene TYPE I1 CELLS IN PRIMATES AND RODENTS diamine (1mg/ml, which protects slides from fading), and examined and photographed in a Leitz Dialux fluorescence microscope with a high pressure HBO 100 mercury lamp and vario-orthomat equipment for photography. PAP technique After preincubation with normal goat serum (1:32 in PBS, pH 7.2) for 15 min a t RT, the lung sections were exposed to SAALS overnight at 4"C, and then successively for 50 min a t RT to goat anti-rabbit IgG (7S, 1 5 0 in PBS), and rabbit peroxidase-antiperoxidase soluble complexes (PAP, 1:64 to 1:1024 in PBS; Nordic Immunological Laboratories, Tilburg, The Netherlands). To enhance the selective type I1 cell staining at early stages of lung development, the latter two steps were repeated (PAP technique according to the double bridge method). Each incubation (30 min) was preceded and followed by rinsing with 2-4 changes of PBS containing 0.1% Tween 20 for 20 min. When dry, the sections were stained for peroxidase (20 min) according to Graham and Karnovsky (1966) and counterstained with methyl green (20 min). After being dipped in alcohol and xylene, the sections were mounted in enthalan. Immunohistochemical controls were performed on the same lung material with preimmunization serum (PS) as the primary antibody or omission of one of the incubation steps. SALS-Hu, SAALS, and PS were used in serial dilutions with PBS (pH 7.2) to a final concentration of 1:400. For the PAP technique, we used PBS containing 5% normal monkey serum. To prevent non-specific background staining in lungs at early prenatal stages, all sera were additionally absorbed (see Ten Have-Opbroek, 1979) with homogenate made from arms of fetal Rhesus monkeys aged 61 or 81 DGA. Carbohydrate Cytochemistry To get more information about the presence of carbohydrates or glycoconjugates in type I1 cells a t early prenatal stages, lung sections (46-81 DGA) were stained with periodic acid Schiff (PAS) or Alcian blue (AB). The nuclei were counterstained with hematoxylin and 0.1% nuclear fast red, respectively. To identify glycogen, some lung sections were exposed to alphaamylase (porcine pancreas; Boerhinger, Mannheim, Germany) prior t o PAS staining. Exposure to 30 p,g amylase/ml solvent for 3 hr resulted in a total loss of PAS staining reactivity. RESULTS lmmunofluorescence When applied to lung sections from adult rhesus monkeys, SALS-Hu (Fig. 1) or SAALS (not shown) yielded a selective, cytoplasmic staining of cuboid cells present in the lining of alveolar ducts and sacs, i.e., type I1 cells. The large and roundish nuclei of type I1 cells (Fig. 1)were always relatively dark. The remaining lining of the ducts and sacs was dark (Fig. l),indicating that the squamous type I cells present were not immunoreactive. Cuboid cells with a dark, large, and roundish nucleus and a cytoplasmic fluorescence pattern were also observable in the lining of respiratory bronchioles and their outpocketings (not shown; 95 see Fig. 4). By contrast, the remaining bronchiolar lining-viz., pseudostratified columnar epithelium consisting of ciliated and nonciliated cells with oblong nuclei, and basal cells-was always devoid of staining. Accordingly, we never found any staining in pseudostratified columnar epithelium lining (sub)terminal bronchioles or bronchi. The usual absence of a fluorescent extracellular lining layer along alveolar lumens (Fig. 1) indicated that the surfactant lining had been removed during tissue processing. Sometimes, surfactant particles that remained behind (Fig. 1) caused some punctate or diffuse staining over the alveolar septa. By contrast, when more or less preserved by a special fixation (heating), extracellular lining layers along alveolar lumens in adult or postnatal lungs (Fig. 2) stained very strongly, indicating that they contained SP-A. In postnatal lungs, the prospective (rather smooth-walled) alveolar ducts and sacs (Fig. 3) were lined by fluorescent cuboid cells with large and roundish nuclei, dark zones (which likely contain mature type I cells, see above), and in places also by thinner fluorescent cells. As reported for other species, the latter cells presumably are intermediate stages in the development of squamous type I cells from cuboid type I1 cells (Ten Have-Opbroek, 1979). Like the prenatal parenchyma (see below), the postnatal parenchyma (Fig. 4) exhibited also small tubules with barely or no visible lumens and a lining of fluorescent cuboid cells with large, roundish nuclei. Structures of these kind (lining: type I1 cells) have been termed acinar tubules or buds (Ten Have-Opbroek, 1979, 1981), because they are the basic structures from which all components of the pulmonary acinus derive, as has been shown for the mouse (Ten Have-Opbroek, 1979, 1981, 19911, the rat (OttoVerberne and Ten Have-Opbroek, 1987), and the human (Otto-Verberne et al., 1988). In the postnatal (Fig. 4) as in the adult lungs, the lining of respiratory bronchioles and their outpocketings contained cuboid cells with dark, large and roundish nuclei and a cytoplasmic fluorescence. Likewise, the bronchiolar linings composed of pseudostratified columnar epithelium (Fig. 4) were never immunoreactive. Alveolar macrophages present in alveolar outpocketings of respiratory bronchioles or on bronchiolar surfaces (Fig. 4) were usually brightly fluorescent, which suggests that they internalize SP-A containing particles from nearby or more distally located extracellular lining layers. Routine inspection of prenatal lungs revealed that at 46 DGA the branching tubular system of the lung was lined exclusively by pseudostratified or simple columnar epithelium. Systems of this kind represent the primordial system of the mammalian lung (Ten Have-Opbroek, 1979,1981,1991).Application of SALS-Hu (Fig. 5) or SAALS to sections did not result in any staining of columnar epithelial (primordial) cells. Occasionally, some fluorescent particles were seen at luminal surfaces, probably caused by artificial retention of conjugate between epithelial ruffles. Mesenchyme (Fig. 5), blood vessels, and pleura (not shown) were not immunoreactive. By 62 DGA, the branching tubular system of the developing lung had a different appearance and consisted of two different parts: a proximal part with pseudostratified or simple columnar epithelium, and a distal part lined by a low-columnar to cuboid cell type with a large and roundish nucleus. As shown for other 96 A.A.W. TEN HAVE-OPBROEK AND C.G. PLOPPER Figs. 1-4. Immunocytochemistry of adult and postnatal rhesus monkey lungs using SALS-Hu (rabbit anti human SP-A) according to the indirect immunofluorescence technique. Fig. 1. Adult lung, alveolar ductslsacs. Cuboid type I1 cells show specific SP-A reactivity (i.e., a unique cytoplasmic fluorescence pattern) that contrasts strongly with their dark, large and roundish nuclei. Squamous type I cells are not immunoreactive. Surfactant (SPA), which remained behind after tissue processing, causes some punctate or diffuse fluorescence over the alveolar septa. X 580. Fig. 2. 9 dPN, future alveolar ductslsacs. The (specifically preserved) extracellular lining layer of these, still smooth-walled, structures displays a bright fluorescence, indicating that it contains SP-A. x 210. Fig. 3. 6 dPN, future alveolar ductslsacs. The epithelial lining consists of cuboid cells having a large, roundish nucleus and a cytoplasmic fluorescence (=type 11 cells), fluorescent bands (=intermediate stages between type I1 and type I cells, tiny arrows), and dark areas ( = mature type I cells). x 220. Fig. 4. 6 dPN, respiratory bronchiole (RB) opening into a future alveolar duct (AD). The lining contains some cuboid cells with a cytoplasmic fluorescence pattern (tiny arrows), i.e., SP-A containing type I1 cells. The pseudostratified columnar epithelium of RB never shows any immunoreactivity. Alveolar macrophages (M) present on the surface of RB or in alveolar outpocketings are usually strongly positive, probably due to uptake of SP-A from the air spaces. The lung parenchyma contains many acinar tubules or buds lined by type I1 cells only (arrowhead) (see Figs. 6-8, 12). x 410. TYPE I1 CELLS IN PRIMATES AND RODENTS Figs. 5-8. Immunocytochemistry of early prenatal rhesus monkey lungs using SALS-Hu (rabbit anti human SP-A) according to the indirect immunofluorescence technique. Fig. 5.46 DGA. The lining of the original branching tubular system, the primordial system of the lung (P),is pseudostratified or simple columnar, and is not immunoreactive. x 390. Fig. 6. 62 DGA. The distalmost branches of the prospective respiratory tract are lined by low-columnar or cuboid cells, with a large and roundish nucleus, that exhibit a prominent fluorescence in apical (andlor basal) cytoplasmic areas (ie., SP-A containing type I1 cells). These branches are the basic structures in pulmonary acinus development, and are termed acinar tubules (A). There is always a n abrupt transition between the alveolar epithelium of the acinar tubule 97 (=fluorescent, cuboid) and the bronchial epithelium of the terminal bronchiole (TB)(dark, columnar). x 330. Fig. 7.81 DGA, terminal bronchiole (TB) opening into three acinar buds. The cuboid or low-columnar type I1 cells constituting these buds frequently display a bright fluorescence throughout the cytoplasm. x 380. Fig. 8. 81 DGA, lung parenchyma containing a dark terminal bronchiole (TB) with a fluorescent acinar bud, a pulmonary artery branch (PA), and some fluorescent acinar tubules (A). The light-emission intensity may be different in places but all cuboid (type 11) cells lining the acinar tubules show a cytoplasmic immunoreactivity for SP-A. x 380. 98 A.A.W. TEN HAVE-OPBROEK AND C.G. PLOPPER mammals, these parts represent the prospective bronchial system and pulmonary acinus, respectively. (Ten Have-Opbroek 1979, 1981, 1991). Specific staining for SP-A (Fig. 6) was also first detectable by 62 DGA, and was restricted to the low-columnar or cuboid epithelium lining the distalmost tubules or buds (termed: acinar tubules or buds, see above). The fluorescent staining was initially mainly observable along the apical (Fig. 6) and/or basal cell borders but was soon found throughout the cytoplasm (Figs. 7, 8). All of the lowcolumnar or cuboid cells displayed SP-A specific fluorescence, beit with a widely varying intensity. This latter phenomenon hampered optimal recording of the overall staining pattern in photomicrographs. There was always a sharp demarcation (Figs. 6, 7) between the distal and proximal parts of the branching tubular system in the developing lungs (criteria: approximately cuboid epithelium with large and roundish nuclei; presence of SP-A reactivity versus pseudostratified or simple columnar epithelium with oblong nuclei; absence of SP-A reactivity). By 81 DGA, the distal part of the prospective respiratory tract consisted of one (Fig. 7) to five (not shown) generations of fluorescent tubules or buds (acinar tubuledbuds). After application of SALS-Hu, fluorescence was most striking in the distalmost buds or tubules; SAALS yielded a more equal staining over all generations, which may be caused by its reactivity to two type I1 cell proteins, namely, SP-A and a 12 kD protein (Van Hemert e t al., 1986). As in earlier stages, SALS-Hu or SAALS never provided any staining of connective tissue or blood vessels (Fig. 8). In older rhesus monkey fetuses (84-95 DGA), lowcolumnar or cuboid fluorescent cells still were the only type of epithelial cells to line the distal part of the prospective respiratory tract. After 95 DGA, we observed locally a more squamous (instead of cuboid) fluorescence pattern and after 128 DGA sometimes also a total absence of staining, a pattern as found in neonatal lungs (see Fig. 3). As mentioned above, these images presumably reflect the presence of differentiating or mature type I cells. Immunohistochemical controls using PS instead of SALS-Hu or SAALS, and other controls were always negative. lamina of larger or smaller tubules of the original branching tubular system of the lung (primordial system). The pseudostratified or simple columnar (primordial) epithelium was almost or fully devoid of staining. By 62 DGA (Fig. 9), a striking positive reaction was observable in the low-columnar or cuboid cells lining the distal branches of the prospective respiratory tract (acinar tubules). The corresponding basal laminas were also frequently positive. Columnar epithelial cells with oblong nuclei lining more proximal branches (terminal bronchioles) showed some positive reactivity (Fig. 9). In even more proximally located branches, i.e., (pre)terminal bronchioles, on the contrary, the staining was usually restricted to the basal lamina (not shown). After treatment with PAS, primordial epithelial cells at 46 DGA exhibited positive (pink or red) granules in their cytoplasm (not shown). Low-columnar or cuboid cells present by and after 62 DGA (Fig. 10) displayed a highly prominent (red) staining in their apical and/or basal cytoplasmic areas. The pattern or intensity of staining was not influenced by cellular height or location. Terminal bronchioles (Fig. 10) usually showed a less pronounced staining of their lining epithelium. The regular finding of PAS positive material in lumens (not shown) suggested that lack of PAS reactivity in epithelial cells (Fig. 10) was caused by artificial loss of intracellular substance. The PAS reactivity in all epithelial cells could completely be blocked by pre-treating sections with alphaamylase. DISCUSSION Type I1 Cell Differentiation and Function Our studies in adult and newborn rhesus monkeys show that type I1 cells present in the lining of peripheral air spaces contain surfactant protein A (SP-A, 35 kD). Type I cells, which occupy the remaining lining of these air spaces, do not comprise this substance. Pseudostratified columnar epithelium with ciliated and nonciliated cells lining terminal and respiratory bronchioles is also devoid of SP-A. The fact that distal nonciliated columnar epithelial cells lack SP-A is not surprising because they have been shown to be mucous cells in the rhesus monkey (Tyler and Plopper, 1985; Plopper et al., 1989; Plopper, 1990) as well as in huPAP technique mans (Ten Have-Opbroek e t al., 1991). The correspondApplication of SAALS to prenatal lung sections ac- ing columnar secretory cells in rodents, also termed cording to the PAP technique yielded similar pictures Clara cells, have a different phenotype and do express a s observed in our immunofluorescence studies. There SP-A, being mainly postnatally and in their apical dowas a prominent cytoplasmic staining of low-columnar main (Oomen et al., 1990; Ten Have-Opbroek, 1991; or cuboid cells lining the distal generations of the pro- Ten Have-Opbroek and DeVries, 19911, which suggests spective respiratory tract (acinar tubules/buds). Be- that they internalize this substance from the air spaces tween 62 and 106 DGA, the distalmost cells in these after birth. The present finding of SP-A containing linings were usually low-columnar, and the more prox- type I1 cells in respiratory bronchioles is not uneximal cells cuboid (see Fig. 10); after 106 DGA, the di- pected in view of the usual presence of type I cells (destalmost cells frequently were also cuboid. These tem- rived from type I1 cells) in their lining (present study; poral differences in height-also observed in our Tyler and Plopper, 19851, and has also been reported immunof luorescence studies-had little or no inf lu- for humans (Ten Have-Opbroek et al., 1991), dogs (Ten ence on the SAALS-immunoreactivity. Application of Have-Opbroek et al., 1990a1, and mice (Ten Have-OpPS to prenatal lung sections and other controls did not broek, 1986). Type I1 cells in the rhesus monkey prove to exhibit the same selective ( =cytoplasmic) staining result in any staining of epithelium or other tissues. pattern for SP-A as type I1 cells in other species such as Carbohydrate cytochemistry the mouse (Ten Have-Opbroek, 1975, 1981, 1991; After application of AB to lung sections at 46 DGA, Oomen et al., 19901, the rat (Otto-Verberne and Ten a positive (light blue) staining was found a t the basal Have-Opbroek, 19871, the human (Otto-Verberne et TYPE I1 CELLS IN PRIMATES AND RODENTS Figs. 9-1 0. Carbohydrate cytochemistry of early prenatal rhesus monkey lungs. (Fig. 9), AB staining; (Fig. lo), PAS staining without pretreatment with alpha-amylase. Fig. 9. 62 DGA, terminal bronchiole (TB, tangentially sectioned) opening into acinar tubules (A), one of which ends into a saccule (S). The low-columnar or cuboid type I1 cells of the acinar tubules (and the basal laminas) contain acid glycoconjugates (light blue staining, arrow), which suggests synthesis of extracellular matrix components. x 300. al., 1988, 1990, 1991; Ten Have-Opbroek et al., 19911, and the dog (Ten Have-Opbroek et al., 1990a). As appears from our present studies in rhesus monkey fetuses, type I1 cells are first detectable in developing lungs by 62 DGA, i.e., in the pseudoglandular period of lung development. The cells are identifiable by means of “adult” distinctive features, namely, an approximately cuboid cell shape, a large and roundish nucleus, and a selective, cytoplasmic staining for SP-A. The initial restriction of SP-A pools to apical or basal cell borders can possibly be explained by the fact that rough endoplasmic reticulum seems to concentrate in those (relatively glycogen-free) peripheral cytoplasmic areas at early developmental stages as has been shown for the mouse (Ten Have-Opbroek et al., 1988) and humans (Otto-Verberne et al., 1988). SP-A expression of type I1 cells in developing rhesus monkey lungs appears not to be influenced by initial differences in cellular height (low-columnar or cuboid), an observation also made in mice (Ten Have-Opbroek, 1979, 1991; Oomen et al., 19901, rats (Otto-Verberne and Ten Have-Opbroek, 1987), and humans (Otto-Verberne et al., 1988, 1990). However, as shown by the varying fluorescence intensity, individual type I1 cells may dif- 99 Fig. 10. 73 DGA, terminal bronchiole (TB,tangentially sectioned) opening into acinar tubules (A). Glycogen (red, =dark staining) predominates in the epithelium of the prospective pulmonary acinus, where it may serve as a substrate for the synthesis of surfactant and other lipids (Ten Have-Opbroek et al. 1988, 1990).As shown by some acinar tubules (asterisks), type I1 cells easily lose their glycogen during tissue processing. PA, pulmonary artery branch. x 260. fer with respect to the level of SP-A expression. In other words, rhesus monkey type I1 cells are probably able to express one of their specific functions, synthesis of SP-A, from the time of their appearance (62 DGA) onward. Similar conclusions have been drawn from immunohistochemical studies in some other species, i.e., the mouse (14.2 DGA), the rat (16 DGA), and the human (70-84 DGA) (reviews by Ten Have-Opbroek et al., 1988,1990b). Further evidence for an early onset of surfactant protein synthesis in humans has been provided by the finding of mRNA coding for SP-A in human fetuses aged 17-18 weeks (Otto-Verberne et al., 1990). Our general conclusion for normal mammalian (adult, fetal) type I1 cells-i.e., that phenotype (lowcolumnar or cuboid cell shape; large and roundish nucleus) and function (SP-A synthesis) come to expression at about the same time-has proved to be true for type I1 cells of alveolar or papillary tumors in mice (Rehm et al., 1988) and dogs (Ten Have-Opbroek et al., 1990a) as well. This general statement is fully compatible with our observations in the rhesus monkey that phenotype and SP-A expressions may get lost concurrently when type I1 cells transform into type I cells. Similar findings 100 A.A.W. TEN HAVE-OPBROEK AND C.G. PLOPPER LUNG DEVELOPMENT FORMATION OF THE PULMONARY ACINUS IN MAMMALS I I 1 1 1 1 Foregut - Trachea acinar tubule sprout GROWTH Primordial system derivative structures acinar tubule @ 12 Figs. 11-1 2. Rhesus monkey lung development follows the pathway proposed for mammals (Ten Have-Opbroek, 1981). Fig. 11. The two lung buds arising from the primitive foregut (in the rhesus monkey, by 27 DGA) develop into the primordial systems of the right and left lungs. The primordial tubules are lined by pseudostratified or simple columnar, primordial epithelium. The development of the primordial system (in the rhesus monkey, between 27 and 62 DGA) takes place in the first part of the pseudoglandular period, which lasts in the rhesus monkey from ca. 27 to 95 DGA. have been reported first for the mouse (Ten Have-Opbroek, 1979), and later also for the rat (Otto-Verberne and Ten Have-Opbroek, 1987) and the human (OttoVerberne et al., 1988). The present investigations on carbohydrate cytochemistry in rhesus monkey fetuses suggest a n early onset of some other type I1 cell functions as well. First, by and after 62 DGA, type I1 cells display a n abundance of glycogen, which among other things has been shown specifically to provide substrates for surfactant lipid biosynthesis and to play a distinct role in prenatal multilamellar body formation (see introduction, and Ten Have-Opbroek et al., 1988, 1990b). The present finding of a n abundance of glycogen in type I1 cells at that time is in agreement with ultrastructural observations in peripheral lung areas in the rhesus monkey great alveolar (type II) cell or precursor cii3.- small alveolar (type 1 ) cell or precursor Fig. 12. Differentiation of the primordial system into the prospective bronchial and respiratory systems starts in the second part of the pseudoglandular period, that is, in the rhesus monkey by 62 DGA. The basic structure in the development of the respiratory unit, the pulmonary acinus, is the acinar tubule or sprout (lining: type I1 cells). The pulmonary acinus grows by budding of type I1 cells. In the canalicular and following periods, type I1 cells lining acinar tubules may develop into type I cells (that is, in the rhesus monkey, by 95 DGA). By this, acinar tubules transform into derivative structures (alveolar ducts, sacs, pouches). The abrupt demarcation between alveolar (cuboid, and later also squamous) epithelium and bronchial (columnar) epithelium is always evident. (Tyler et al., 1988, 1989), and also with those in the mouse (Ten Have-Opbroek et al., 1988,1990b) and the human (Otto-Verberne et al., 1988) at comparable stages. Second, by and after 62 DGA, type I1 cells contain acid glycoconjugates, which suggests that the cells may synthesize basal lamina components. Primary Role of Type I1 Cells in Pulmonary Acinus Development The present study indicates that the prospective respiratory system, and thus its units, the pulmonary acini are first detectable in developing rhesus monkey lungs by 62 DGA. The respiratory system, and the more proximally located prospective bronchial system, develop at that time from the undifferentiated primordial system present (see interpretive diagrams for 101 TYPE I1 CELLS I N PRIMATES AND RODENTS TABLE 1. Aspects of lung development in primates and rodents in relation to gestation time' Human First appearance Lung primordium Type I1 cells Term DGA 25 77 265 Rh-monkey % 9 29 100 DGA 272 62 165 Rat % 16 38 100 DGA 10.5 16 22 Mouse YO 48 73 100 DGA 9.5 14.2 19 YO 50 75 100 'Expressed in days of gestational age (DGA), and as a percentage of total gestation time. For details on other developmental data, see present study; Ten Have-Opbroek (1981, 1991); Otto-Verberne and Ten Have-Opbroek (1987); Otto-Verberne et al. (1988). 'Based on photographs of embryos (Heuser and Streeter, 1941). mammalian lung development, Fig. 11,12). This major developmental step takes place in the middle of the pseudoglandular period of lung development, which lasts in the rhesus monkey from approximately 27 DGA (time of appearance of the lung primordium) to 95 DGA (see below). Our present study further shows that, also in the rhesus monkey, type I1 cells play a key role in pulmonary acinus formation (see Fig. 12). First, between 62 and 95 DGA (i.e., in the last part of the pseudoglandular period), type I1 cells are the only cells to line and generate the prospective pulmonary acinus and its components, the acinar tubules and buds. Second, after 95 DGA (ie., in the canalicular, terminalsac, and alveolar periods), type I1 cells also give rise to a flatter cell type in the lining of the prospective pulmonary acinus, i.e., precursors of type I cells. This observation is in line with our earlier ultrastructural observations in rhesus monkey lungs, i.e., low-cuboid cells line shallow depressions in prospective respiratory bronchioles (Tyler et al., 1988). Third, also in later prenatal and postnatal stages of lung development, proliferation and budding of type I1 cells seem to be responsible for the final maturation of the pulmonary acinus including alveolarization. A comparable developmental pattern-proliferation and budding of epithelial cells from the tracheal lining into the underlying mesenchyme-has been reported for rhesus tracheal submucosal glands (Plopper et al., 1986b). Our present conclusion concerning a primary role of type I1 cells in pulmonary acinus formation in the rhesus monkey fully agrees with our findings in some other species, i.e., the mouse (Ten Have-Opbroek, 1979, 1981,1991; Oomen et al., 19901,the rat (Otto-Verberne and Ten Have-Opbroek, 1987), and the human (OttoVerberne et al., 1988,1990). In summary, we conclude that the histologic pattern of lung development in the rhesus monkey corresponds with the patterns found for rodents and humans (reviews by Ten Have-Opbroek, 1981, 1991; Ten Have-Opbroek et al., 1988). Timing of Lung Development in Primates and Rodents The timing of lung development in primates and rodents shown in Tables 1and 2 is based on data from the literature (Heuser and Streeter, 1941) and our own data (see Table 1). Comparison of the time course of lung development in rhesus monkeys and humans on the one hand and mouse and rats on the other hand reveals (Table 1) that the timing of type I1 cell differentiation is different for primates and rodents, i.e., at 29-38% and 73-75% of the total gestation time, respectively. However, as shown in Table 1, the lung primordium appears in these two mammalian classes also at different times (at 9-16% versus 48-50% of gestation). Taking this into account, the onset of type I1 cell differentiation in primates and rodents seems to take place after a more or less similar developmental interval (20-25% of the total gestation time). As reported (Ten Have-Opbroek, 1981), this major interval covers the establishment of the primordial system. The second major developmental interval (Table l),which extends from the onset of type I1 cell differentiation to term, covers the prenatal development of the respiratory and bronchial systems (Ten Have-Opbroek, 1981). As appears from Table 1, this specific period does show distinct species differences. In primates it lasts about twice as long as in rodents (62-71% versus 25-27% of the total gestation time). More accurate information on the issue of species differences can be obtained (Table 2) by relating the duration of the two major specific developmental periods to the total time covering prenatal lung development. In this light, establishment of the bronchial and respiratory systems proves to last in primates about 1.5 times longer than in rodents (75-78% versus 5142%). For the development of the primordial system it is of course just the reverse (22-25% versus 48-49%). Possible mechanisms for the shorter duration of this period in primates might be the following. First, a higher growth rate of primordial epithelium might enable primates to accomplish a primordial system comparable in size to that in rodents within a shorter time. Second, this formation is terminated at an earlier time-point by a faster development of the peripheral capillary system in primates. Proximity to primordial epithelial cells possibly promotes their differentiation to type I1 cells (Ten Have-Opbroek et al., 1988). Finally, and maybe most likely, the development of the bronchial and respiratory systems does not need a primordial system with a mammalian-specific critical mass. Anyhow, the longer duration of the second specific developmental period in primates is not surprising in view of the complexity of the final bronchial and respiratory systems (length and number of generations of branches) in higher mammals. In the evaluation presented here, we have left the period of postnatal lung development out of consideration for clarity reasons. This period deals only with final structuring and growth of the already existing and functioning bronchial and respiratory systems. Moreover, it is an extremely long period, because it virtually extends t o the end of the puberal or growth period. Although the bronchial system starts to develop at the same time as the respiratory system in the rhesus 102 A.A.W. TEN HAVE-OPBROEK AND C.G. PLOPPER TABLE 2. Duration of specific periods in lung development in primates and rodents’ Human Duration Prenatal lung development2 Primordial system development3 Bronchial and respiratory systems development4 Rh-monkey DGA % DGA % 240 52 100 22 138 35 100 25 188 78 103 75 Rat Mouse DGA % DGA % 11.5 5.5 100 48 9.5 4.7 100 49 6 52 4.8 51 ‘Expressed in DGA, and as a percentage of the total time of prenatal lung development. 2Calculated from the time of appearance of the lung primordium until term (see Table 1). 31nterval between the detection of the lung primordium and the appearance of type I1 cells (see Table 1). 41nterval between the appearance of type I1 cells and term (see Table 1). monkey, i.e., by 62 DGA (present study), the proper process of bronchial epithelial differentiation in the respiratory tract as expressed by the appearance of ciliated cells starts earlier, that is, in the trachea by 46 DGA (Plopper et al., 1986a). After that time, bronchial epithelial differentiation proceeds in a distal direction throughout the airway tree (Tyler et al., 1989; Plopper, 1990). CONCLUSIONS Our conclusion that type I1 cell differentiation and function in the rhesus monkey (Macaca mulatta) start by 62 DGA, that is, in the pseudoglandular period of lung development fully agrees with others’ observations in the macaque. In the same species (Macaca muZatta) (Kerr et al., 1975), the cells lining the peripheral tubules of the developing lung become cuboid in height from the 50th to 75th day of gestation, when the “glandular” pattern is still characteristic. At the same time, their cellular organelles increase in number, short microvilli and terminal bars become obvious a t their apical surfaces, and capillaries with thickened endothelial cells come to lie in close proximity. In the pig-tail monkey, Macaca nemestrzna (Boyden, 19761, which has a similar gestation time (about 168 days), peripheral rosettes of epithelial buds destined to develop into the respiratory units of the lung (Boyden, 1976) are first noticeable about the 57th gestational day; their epithelium is made of closely packed cuboid cells. Our conclusion for the rhesus monkey that type I1 cells start to give rise to type I cells by 95 DGA, is also supported by these studies in the macaque. As reported, flatter cells occur in the originally cuboid epithelial lining of peripheral tubules by 100 (Kerr et al., 1975) or at 106 (Boyden, 1976) days gestational age and thereafter. The underlying mechanism, capillary invasion of overlying cuboid cells, starts by the gestational age of 75 days in the rhesus monkey (Kerr et al., 1975), and 80 days in the pig-tail monkey (Boyden, 1976).However, a possible alveolar nature of the cuboid cells is never discussed (Kerr et al., 1975; Boyden, 1976).On the contrary, it has generally been thought for a long time that cuboid epithelium in developing and adult mammalian lungs represents (prospective)bronchial epithelium (reviews by Ten Have-Opbroek, 1981; Ten HaveOpbroek et al., 1991). However, by contrast, it is not feasible to relate our time-points of type I1 or type I cell differentiation in the rhesus monkey (and other mammalian species) to timepoints of other investigators in rodents for the following reasons. One group of investigators reports not on the first appearance of type I1 cells but on the expression of a differentiated type I1 cell function at the end of gestation such as a peak in alkaline phosphatase activity (Edelson et al., 1988), adult changes in the pattern of cytokeratin synthesis (Paine et al., 19881,or correlations between membrane and secretory differential markers like cell-surface glycoconjugates and surfactant phospholipids (Joyce-Brady and Brody, 1990), or provides quantitative ultrastructural data on type I1 cell differentiation a t late gestational ages (Lin and Lechner, 1990; Snyder and Magliato, 1991; Young et al., 1991). A second group of investigators, on the other hand, does report on the first appearance of type I1 or type I cells based on antigenic or biochemical marker studies in developing mouse or rat lungs. However, the relation between markers and specific epithelial cell types in developing lungs is not or not sufficiently explained. Studies on mouse lung development (Jaskoll et al., 1984, 1988; Slavkin et al., 1989) conclude that type I1 cells first appear at 16.5 DGA in vivo, that is, a t the beginning of the canalicular period. This conclusion is based on the detection of SP-A expression in a number of cuboid cells at that time. However, morphologically identifiable (and SP-A reactive) type I1 cells are present in developing mouse lungs as early as embryonic day 14.2, and are the only cells to line the future respiratory unit a t earlier stages (Ten Have-Opbroek, 1975, 1979, 1991; Ten Have-Opbroek et al., 1988, 1990b). The relatively late detection and the small number of reactive type I1 cells in developing and adult lungs (Jaskoll et al., 1984, 1988; Slavkin et al., 1989) suggest that only a subpopulation of mouse type I1 cells has been stained by the antibody used (anti human SP-A; source: human alveolar proteinosis lavage material). Studies on rat lung development using antigenic or biochemical cell markers, provide some different time-points for the first appearance of type I1 or type I cells. Surprisingly, only one study (Funkhouser et al., 1987) relates labeling to cell morphology. This study using a monoclonal antibody to the luminal surface of rat type I1 cells (JBR-1, Funkhouser et al., 1987) detects labeling on or in cuboid cells as early as 14 DGA, that is two days earlier in the pseudoglandular period than we reported (Otto-Verberne and Ten HaveOpbroek, 1987). The labeled structure (Funkhouser et al., 1987) was found at a level caudal to the tracheal bifurcation and ascribed to staining of the epithelial lining of the two embryonic main stem bronchi. The authors conclude that progenitors of type I1 cells at that level already express the JBR-1 reactive type I1 TYPE I1 CELLS IN PRIMATES AND RODENTS cell antigen by day 14. As mentioned but not shown (Funkhouser et al., 19871, the antigen continued to be expressed by cuboid cells lining the proximal embryonic respiratory ducts between 14 and 18 DGA, and finally appeared on selected cells in the smallest ducts by 19 DGA. However, there is some evidence that the JBR-1 antibody initially reacts to endothelium of embryonic pulmonary arteries, and later also to a subpopulation of type I1 cells. First, unlike the embryonic main bronchi (lining: pseudostratified columnar epithelium with oblong nuclei, Ten Have-Opbroek, 1981, 19911, embryonic arteries have a prominent cuboid endothelial lining. Second, as shown by the ultrastructural studies in adult rat lungs (Funkhouser e t al., 19871, some endothelial surfaces do react to JBR-1. Third, double labeling studies which we performed with JBR-1 and anti SP-A a t 19 DGA (unpublished observations) detect a distinct JBR-1 reactivity (cuboid fluorescent cells) in pulmonary artery branches accompanying bronchioles and sometimes also in smaller vessels, whereas the cuboid epithelium of prospective respiratory structures (present from 16 DGA onward and consisting of type I1 cells, Otto-Verberne and Ten Have-Opbroek, 1987) always labels with anti SP-A but only infrequently with JBR-1. This infrequent labeling of type I1 cells by JBR-1 at 19 DGA-also observable in photomicrographs of adult rat lungs (see Funkhouser et al., 1987)-indicates that JBR-1 reacts to a subpopulation of type I1 cells by the end of gestation. In other words, the monoclonal antibody JBR-1 does not provide a suitable marker for studies on type I1 cell differentiation. The antigenic determinants recognized do not represent a consistent feature of type I1 cells, and are not type I1 cell specific. Other studies in rats also conclude t h a t type I1 cells start to differentiate in the pseudoglandular period but at a later time (on 16 DGA; Williams and Dobbs, 1990; Woodcock-Mitchell e t al., 1990). Type I cells are said to be first detectable in the same period, by 15 DGA (Williams and Dobbs, 1990). The reagents used for specific cell detection are the lectin Muclura pomiferu, MPA (Williams and Dobbs, 1990) or antibodies to keratin No. 18 (WoodcockMitchell et al., 1990) for type I1 cells, and antibodies to a n unknown antigenic determinant for type I cells (Williams and Dobbs, 1990). As mentioned (Williams and Dobbs, 1990), MPA binding is detectable indeed on 16 DGA and thereafter but, as shown in the illustrations, it has a widespread distribution. The authors do not correlate this labeling to the specific lung cell types present. Criteria that can be used for this purpose-i.e., the shape and size of individual epithelial cells and their nuclei, and the arrangement as a simple or pseudostratified epithelium (Ten Have-Opbroek et al., 1988, 1990b)-are not optimally visualized in the photomicrographs (see Williams and Dobbs, 19901, due to a high background staining in the fluorescence pictures and the use of phase contrast images for routine orientation. Despite this fact, however, i t is evident that by and after 16 DGA MPA binding occurs in cells scattered in the lining of larger tubules with pseudostratified or simple columnar epithelium (i.e., bronchial epithelium) and smaller peripheral tubules with approximately cuboid epithelium (i.e., alveolar epithelium consisting of type I1 cells), see Otto-Verberne and Ten Have-Opbroek, 1987. Because the labeling pattern 103 of the peripheral tubules is discontinuous, and even smaller structures like acinar buds ( = newly formed type I1 cells) seem to be devoid of staining (see illustrations in Williams and Dobbs, 19901, MPA binding apparently involves a subpopulation of type I1 cells, i.e., probably the more mature cells that occur more proximally and have a more or less cuboid shape. As appears from the pictures, antibodies to keratin No. 18 (Woodcock-Mitchell et al., 1990) also label both bronchial epithelial and type I1 cells in developing lungs. The monoclonal antibody for type I cells has a widespread reactivity as well (see photomicrographs in Williams and Dobbs, 1990). On and after 16 DGA, this antibody seems to bind to bronchial epithelial cells and a subpopulation of type I1 cells; by 15 DGA, i t seems to react occasionally to the columnar epithelium of the original branching tubular system of the lung, the primordial system (cf. Otto-Verberne and Ten Have-Opbroek, 1987). In the adult rat lung (Dobbs et al., 19881, there is also some reactivity with type I1 cells. In summary, our conclusions for the rhesus monkey (and some other mammalian species) concerning the first appearance of type I1 or type I cells cannot be compared to recent conclusions for rodents, because the markers used for cell identification in the rodent studies lack the required ( = unique and persistent) cell specificity. However, our conclusions fully agree with others’ findings in the macaque. ACKNOWLEDGMENTS The authors thank Mr. Erwin C. P. de Vries and Mrs. Alison J . Weir for technical assistance, Mrs. Joyce W. Wetselaar for making the explanatory drawings, Mr. Jan H. Lens for photographic assistance, and Mrs. E. A. van der Kwast and Mrs. G.N. Augustina for secretarial assistance. This work was supported in part by Public Health Service grants HD 24959 and HL 28988, and the Netherlands Asthma Foundation. 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