Pulmonary Perspective Matrix-driven Pneumocyte Differentiation 1- 3




Statement of the Problem A normal adult mammalian alveolar epithelium is a mosaic consisting of type I and type II pneumocytes with a numerical ratio that' is constant. The two cell types serve distinct functions (1). Whereas in adult lungs type II cells are believed to be precursors for type I cells(2, 3), it has not been established whether in fetal lungs type II cells are obligatory precursors of type I cells, or whether there is a common precursor for the two cell types (4). Type II cells are frequently observed at alveolar comers. During repair, there is a transient increase in type II cells. Differentiation of daughter type II cells into type I cells restores the numerical ratio of type II to type I cells and their normal localization. Three fundamental questions result from these observations. (1) What factors determine the localization of type I and type II cells? (2) How is the constant ratio between type I and type II cells in adult lungs maintained? (3) When, during lung development, are the patterns of type I and type II celllocalization established? In this commentary I will propose a hypothesis that provides a working framework for understanding how the cell patterns of a normal alveolar epithelium may be established and restored after injury. Lung basement membrane may playa key role in determining the location of type I or type II cells in the lung. The extracellular matrix effects are mediated via specific cell surface receptors (5, 6). Extracellular matrix effects with specific reference to the lung have been recently reviewed (7-9). Background The primary function of the lung is to permit efficient gas exchange between venous blood and alveolar gas. This function is accomplished by an architecture in which the thinnest possible tissue barrier consisting of type I epithelium and capillary endothelium are separated by a composite epithelial and endothelial basement membrane across which gas exchange occurs. Although the two basement membranes appear fused, they can be distinguished under certain conditions (10, 11). Along the alveolar septa, thin segments alternate with thick regions in which the endothelial and epithelial basement membranes are separated by interstitial components. Perhaps a better appreciation of this can be 452

SUMMARY ThJscommentary presents evidence In support of a hypothesis that adult mammalian alveolar epithelial basement membrane possesses functional and structural domains that determine sites at which type I and type II cells localize. The hypothesis provides a framework for understanding how, after normal repair of the epithelium, a constant ratio of type I and type II cells, and the localization of the cell types Is maintained. AM REV RESPIR DIS 1991; 144:452-457

gained by considering architectural changes that occur during postnatal lung development. Development oflungs in many mammalian species: human (12),rat (13-17), rabbits (18), cats (18),mice (19),is incomplete at birth. Respiratory bronchioles and alveoli are formed postnatally. Stages of postnatal rat lung development are depicted in figure 1. In the newborn rat, large conducting airways with columnar epithelium end in thick-walled saccules that contain a double capillary network (14). In the subsequent 2 wk, branching of secondary septa from primary septa forms angles recognizable in mature lungs as "alveolar corners." The double capillary network is transformed into a singlecapillary network, which is accompanied by thinning of alveolar septa, and results in the alternate thick and thin segments of the alveolar wall. Terminal bronchioles are converted into respiratory bronchioles by out-pouching of the walls and close apposition of the capillary loops to the epithelium. These observations suggest that patterns ofepithelial celllocalization may be established during this period of lung development. A key observation from these studies is that even in the newborn animals, the epithelium over the capillary endothelium is almost as thin as that observed in adult animals (14). Thus, the type II cell phenotype is usually not favored over regions of fused epithelial and endothelial basement membranes. In mature animals, type I cells cover 95070 of the respiratory surface,whereas type II cells cover the remaining 5% (20). Although type I cells are usually depicted as pancakeappearing cells,they havea more complex appearance, with unique functional consequences (13, 21, 22). In a three-dimensional reconstruction, a type I cell appears as a dendritic structure in which a central nucleated plate is connected through thin cytoplasmic stalks to several nonnucleated plates. The different plates may be located on different

alveolar surfaces. Type I cells are believed to be specialized for efficient gas exchange (22, 23). They also provide the major barrier to fluid efflux into the alveolar space. Because of difficulties in isolation of this cell type, we know very little about the biochemical or cellular functions of these cells. In contrast, type II cells have been extensively studied. Studies of these cells initially focused on surfactant production, but more recently other functions of the cells have also been reported (1, 24, 25). In situ, type II pneumocyteshave cuboidal structures. For the purpose of this discussion, I willassume that type II cells are located at alveolar corners, although a systematic study of their localization has not been done. The alveolar epithelium has a stereotypic response to injury from whatever cause (figure 2). After injury, which results in necrosis of type I cells, type II cells proliferate and migrate to the exposed areas of the basement. In some cases, the regenerating type II cell epithelial cells may form a layer several cells thick (26). The type II cells subsequently differentiate and transform into type I cells. In the restored normal lung, the location of type II cells at alveolar corners is maintained, whereas type II cells, which come to reside on other areas 0 f the alveolus,

(Received in original form July 16, 1990 and in revised form February 4, 1991) 1 From the Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut. 2 Supported by Grant HL-41854 from the National Heart, Lung, and Blood Institute. 3 Correspondence and requests for reprints should be addressed to Jamson S. LwebugaMukasa, M.D., Ph.D., Pulmonary and Critical Care Section, Department of Internal Medicine, Yale University School of Medicine, P.O.Box 3333, 333 Cedar Street, New Haven, CT 06510.



tors, hormones, and mechanical factors) are important for cell differentiation, migration, and lung morphogenesis, they do not completely explain the apparent nonrandom distribution of type II cells or their constant numerical ratio to type I cells in normal adult lungs. On the other hand, it has been observed, largely from studies of nonpulmonary systems, that basement membranes may not only regulate cell differentiation but may also determine the sites of its occurrence.

Fig. 1. Stages during postnatal developmentofratlung. Pane/1. Atbirth,large conducting airwayendsin athick-walled saccule containing a double capillary networkand randomlydistributedtype II cells. Pane/2. By the fourth postnatal day the saccule has enlarged, and crestsformingsecondary septahaveappeared. Pane/2a. Enlargement of a portion of Panel2 showingthe major components of the secondary septum. A type I epitheliumrestson a continuous basement membrane; type II cell is at the base (futurealveolarcorner)of the crest(E1C == elastinandcollagenfibers. c == alveolar capillary. IC == interstitial cell). Pane/3. Between Days 4 and 14 the secondary alveolarsepta elongate by backward expansion of the interstitial components capillariesand proliferation of type II cells at the alveolarcorners. The septa still contain a double capillary system. Pane/4. Between Day 14and Day21 there is markedthinning of the alveolar septa, which results in formation of a single alveolarcapillary network with alternatingthick and thin segments separating theepithelium and endothelium.


Fig. 2. Response of alveolar epithelium to injury. Injury to lung epithelium may resultin increasesof type II cells,denudation. and damageto epithelial basementmembrane. Type IIcellsproliferate, migrate,and cover the denuded basement membrane and repair the damaged surfaces. Subsequently, daughter cells differentiate into type I cells, thus restoring normal architecture. Under certain pathologic conditions such as in fibrotic reaction,the epitheliumoverlying plasma clots retainsa cuboidal appearance, presumably becauseof stimulation by serum factors, paracrine, and/or autocrine growth factors.





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are transformed into type I cells. Thus, the normal epithelial cell distribution and a constant ratio between type I and type II pneumocytes are maintained. In addition to their stem cell function, type II cells perform many other metabolic and biochemical functions (1), including synthesis and deposition of basement membrane (24,27-29). Type II cells are also capable of remodeling lung basement membrane (7). During periods of rapid lung growth and repair, discontinuities appear beneath type II cells (30, 31). Type II cells may


also secrete hydrolytic enzymes, which may be active in the alveolar space (32, 33). Lamellar bodies contain lysosomal enzymes, which are secreted in alveolar fluid along with surfactant components. Physiologic functions of the secreted enzymes are unknown at present. Type II cells synthesize,store, secrete,and recyclesurfactant components (4, 25, 34-36). They also play an important role in alveolar fluid electrolyte transport (37-39). Although other factors (cell-to-cell interactions, CAMs, cadherins, soluble growth fac-

Hypothesis A plausible hypothesis to explain both a constant numerical ratio of type I and type II cells and the nonrandom localization of type II cells, away from the thinnest segments of the alveolar septa, is that the lung basement membrane contains functional and structural microdomains that regulate pneumocyte differentiation into either type I or type II pneumocytes and determine the topologic localization of the epithelial cells in the alveoli. The pattern of epithelial cell localization is established during the neonatal period. Basement membrane domains in which there is close apposition 0 f capillary endothelium and alveolar epithelium may be important for transformation of type II into type I pneumocytes. During normal lung repair, epithelial cells over the thinnest segments of alveolar septa become type I, whereasthose at other sites retain type II phenotype, thus reestablishing the normal distribution pattern and numerical cell ratio. The implication of this hypothesis is that during repair, lung basement membrane may not only determine the path of epithelial cell differentiation but may also determine the sites where differentiation can occur. Because of the branching structure of type I pneumocytes, functional response to a given basement membrane domain may be reflected in changes of cytoplasmic plates extending over a wide area, away from the basement membrane region initiating the effect. This would explain why, for example, flattened type I cells are observed over "thick" segments of alveolar septa. Several reported observations support this hypothesis, and they are discussed in detail below. A number of structural domains have been described in the alveolar epithelial basement membranes and correspond to locations of types I and II pneumocytes (figure 3). Vaccaro and Brody (11) showedthat areas beneath type II cells contain discontinuities that increase in number during periods of lung remodeling (figure 3, panel 1). Sannes (40) and van Kuppeveltand coworkers(41) demonstrated that basement membranes beneath type I and type II cells possess distinct sulfated ester compositions (figure 3, panel 2). Proteoglycans may contribute to these differences. Perhaps the most readily recognizable structural domains of the alveolar epithelial basement membrane are the areas in which the epithelial and endothelial basement membranes appear fused. Thus, the alveolar epi-



(1) II


~ 'fused' AM



thelial basement membrane can be thought of as consisting of two major domains (figure 3, panel 3): one in which it is in intimate contact with the capillary endothelial basement membrane, and another in which the two basement membranes are well separated. Such differences may be significant for epithelial cell differentiation and function. Finally, Grant and coworkers (17)and Rosenkrans and Albright (42) reported differences in fibronectin labeling beneath type II cells compared with that beneath type I cells (figure 3, panel 4). These observations provide the structural evidence that the alveolar basement membrane is topologically differentiated. Given the recognized intimate apposition of the epithelial and endothelial cell layers in the thin alveolar septal segments, it is surprising that interactions between lung epithelium and capillary endothelium have not been investigated as possible regulators of pneumocyte differentiation. Observations made in developing human and rat lungs suggest that the endothelium may play an important role in the transformation of terminal bronchioles into respiratory bronchioles and the thinning of the epithelium over capillaries in alveoli (12, 14). The next group of observations show that type II cells are capable of responding in different ways to different basement membranes. Human and rat alveolar extracellular matrices retain their three-dimensional architecture and spatial distribution of the constituent components after removal of all cellular elements with detergents (43). Such matrices can be used as substrata for studying epithelial cell responses to lung basement membranes (43). Our laboratory and those of others haveshownthat isolated type II cells, when cultured on different basement membrane systems, respond differently (27,43-47). In our earlier observation of adult type II cells cultured on human lung matrix, weobserved that flattened cells covered the majority of the lung matrix. On reexamination of the same samples, wefound an occasional cuboidal cell at alveolar corners (unpublished observations). Thus, type II cells may not only respond in specific waysto different basement membranes but may respond differently to structurally different regions of the same basement membranes. However,in vitro studies of basement membrane regulation of pneumocyte differentiation are limited by a number of factors: (1)

Fig. 3. Structural domains of alveolar epithelial basement membrane(AEBM). 1.Discontinues beneathtype IIepithelial cells through which the cells interact with interstitial cells (IC).2. Differences in sulfatedester charge beneath types I and II pneumocytes. 3. Regions in whichepithelialand capillarybasement membranes (CBM)are fused. 4. Differencesin fibronectin distribution beneath types I and II pneumocytes.

adult type II cells do not divide and lose their differentiated features in culture; (2) cell isolation procedures may select subpopulations of type II cells that are already committed to a type I cell phenotype; (3) primary isolations of type II cells may vary in purity, and contaminating nontype II cellsmay confound experimental observations. To overcome some of these problems, we developed a clonal cell line, LM5, which was derived from type II cellsisolated from lO-day-old Sprague-Dawley rat pups (28). The cell line retained a number of morphologic and immunocytochemical features of type II cells and formed homogenous monolayers when cultured on dishes coated with reconstituted mouse tumor (EHS sarcoma) basement membrane (Matrigel and Lwebuga-Mukasa,unpublished observations). In contrast, when LM5 cells were cultured on an acellular rat lung matrix, a mosaic of type I and type II cell phenotypes was observed. It is of interest that the type I-like cells on this substratum most closelyresembled the morphologic features described by Weibel (21), including formation of multiple apical membrane plates. Morphometric studies will be required to determine whether the type 11like cells localize in alveolar corners of the matrix. This study provides the first direct evidence that the alveolar epithelial basement membrane can induce a clonal cell line to differentiate into both type I and type II cell phenotypes. Response of type II cells to a preformed basement membrane requiresadditional comment. Vracko (48) observed that during lung repair after oleic acid lung damage, in areas where lung basement membrane was intact, repair ensued without deposition of new basal lamina. However, in areas where severe damage had occurred, new basal lamina was deposited, and in some cases normal repair did not occur. We have observed in vitro that the nature of the basement membrane deposited by type II cells can be regulated by the basement membrane or its purified components (Lwebuga-Mukasa and others, unpublished observations). Type II cells cultured on human amniotic basement membrane deposited a new basal lamina (27), whereas type II cells cultured on lung basement membrane did not deposit a newbasal lamina (43).When isolated type II cells were cultured on plastic vesselscoated with purified extracellular matrix components, in general, increased basal lamina components weredeposited when the

substratum consisted of interstitial components than when it consisted of basal lamina components (Lwebuga-Mukasa and others, unpublished observations). These observations suggest that lung basement membrane may regulate its own renewal, which may be one of the mechanisms for the maintenance of the distinct structural domains of the basement membrane. How do we then account for the increase in type II cells during repair? The response of the alveolar epithelium to injury is depicted in figure 2. After lung injury, local basement membrane effects may be overcome by influences from cytokines or other soluble growth factors released by inflammatory and parenchymal cells and from serum exudate. As a result, type II cells proliferate, migrate, and repair damaged epithelial basement membrane. As the factors that stimulate pneumocyteproliferation and migration subside, local basement membrane influences may regulate the differentiation of the alveolar epithelium and restore normal cytoarchitecture. Interaction of the alveolar epithelial cells with the capillary endothelial cellsthrough their composite basement membranes may beespecially important for the restoration of the normal alveolar architecture. However, if the injury resultsin severedamage or if the injury is compounded by other as yet unknown factors, the alveolar epithelium fails to return to its normal appearance, and a cuboidal epithelium may be observed. In such cases wewould predict alteration of the distribution of at least some of the functional basement membrane domains. There are examples in other systems (urogenital, musculoskeletal, nervous, and cardiovascular systems) where extracellular matrix components have been shown to determine the locations at which differentiation occurs. In repair of renal tubules (49), and in regeneration of peripheral nerves, basement membranes play key roles in guiding repair processes. In repair of traumatic muscle injury, the basal lamina determines the site of implantation and differentiation of the nerve terminal (50-53). A laminin-like adhesion protein has been shown to be involved in the formation and stabilization of the synapses (53). Using an acellular amnion model system, Davis and coworkers (54) demonstrated that the basement membrane surface guides neurite growth in vitro, and it can serve as a bridge for regenerating neurons in the nervous system in vivo. During embryonic development of the vertebrate heart, the myocardium determines the sites at which endothelial cells are transformed into prevalvular mesenchyme (55,56). The myocardial influence is mediated by adherons, which are fibronectin-containing particulate aggregates that accumulate in the basement membrane between the endothelial and the myocardium at specific sites initiating the induction. These observations provide the strongest evidence for basement membrane components as determinants of the locations at which differentiation may occur.


Localization of type II cells at specific sites may have its origins during the period of definitive alveolar formation (14). As discussed earlier, in humans and in many other mammalian species, definitive alveoli and respiratory bronchioles are formed postnatally (figure 1).Sites where secondary septa branch from primary septa form readily recognized alveolar "corners" (figure 1, panel 2). Type II cells are found at bases of septal crests, the future alveolar corners. The structure of the epithelial basement membranes at sitesof secondary septa formation has not been evaluated using antibodies to specific extracellular matrix components. However, from the electron microscopicdescriptions of Burri (14) and Kauffman and colleagues (16), severalfeatures were constantly observed at sites of secondary septa formation, namely, type I cell epithelium overlying a continuous basement membrane, deposits of collagen and elastic fibers that wereintimately associated with the epithelial basement membrane, capillary endothelial cells, and mesenchymal cells. The alveolar septum growsby the proliferation of interstitial and capillary endothelial cells and type II epithelial cells at the bases of the secondary crests (16). Current understanding of the biology of alveolar epithelial cells suggests that a differentiated phenotype of the cells is best defined by a panel of functional and morphologic features (57). In vitro, components of this panel can be shown to be regulated by combination extracellular matrix components, soluble factors, cell-to-cellinteractions, and mechanical factors. My postulate is that during the neonatal period, the layout of the structural and functional domains of the lung epithelium is determined. This layout provides the boundaries within which normal remodeling may occur and beyond which abnormal . repair ensues. From that point on, the differentiation path taken by a given alveolar epithelial cell (i.e., whether the cell retains type II cell phenotype or transforms into type I cell) depends on the nature of the basallamina it rests on. This interaction between the cell and its basal lamina is dynamic since the cell, in addition to responding to the basement membrane, is also capable of depositing or remodeling the basement membrane. In this context, abnormal repair may occur when the alveolar layout is so damaged that the cells do not have a proper basement membrane template.

Alternative Hypotheses Let us now examine some ofthe reasons why other factors are less likely to be candidates for determining the localization of type II cells.Epithelial-mesenchymalcell interactions have been shown to be important for normal morphogenesis of the lung (58, 59). Epithelial airway buds will divide only if they are in intimate contact with the mesenchyme. It is believedthat undefined mesenchymal factors, which are in part mediated by intervening basement membrane, may mediate this pro-


cess (60-62). During periods of rapid lung ical distortion of the culture substratum has growth and repair, type II cells are seen to been reported to alter secretory and biochemextend processes through discontinuities in ical functions of isolated type II cells (7, 73). basement membrane and interact in some way The transduction of the mechanicalinfluences with interstitial components (30, 31,63, 64). most likely results from interactions of epiIt has not been resolved whether perforations thelial cells as a sheet that is anchored on a beneath type II cells simply represent baseflexible extracellular matrix through cell-toment membrane remodeling by type II cells extracellular matrix receptors. It can be aror are required for transmission of signals gued that variations in stress forces in alveofrom interstitial compartment to the epithelus may in some way favor localization of type lial cells. It has not been determined what fac- II cells at alveolar corners. It is of interest tors would result in localization of such inin this respect that the distribution of type teractions only at alveolar corners. I and type II cells remains unchanged during There are soluble factors that are secreted periods of prolonged atelectasis. by interstitial cells (65, 66) and affect type II cell maturation or type II cell factors that General Proposed Approaches to affect fibroblast growth. The function of the Resolve the Problem former in adult lungs or repair is unknown. The proposed hypothesis suggests a number Leslie and coworkers (67) reported in vitro of testable questions. For example: Do alvestimulation of DNA synthesis in rat type II olar endothelial cells regulate alveolar epithecells by alveolar macrophage supernatants. lial cell differentiation? Are basement memPneumonectomized rabbit serum has been brane microdomains distributed differently reported to contain a somatomedinlike pepin lungs with increased type II cell populatide that stimulates type II cell proliferation tions compared with those of normal lungs? (68). However, since the soluble factors are Do alveolar epithelial basement membranes diffusible, they do not completely explain the at different stages of lung development or relocalization of type II cells.However,it is pos- pair support distinct patterns of epithelial cell siblethat diffusible factors that are active only differentiation? How are basement membrane at a short range, and are secreted by interstidomains established in the neonatal period tial cells fixed at alveolar corners, determine maintained and restored after injury? the location of type II cells. It appears, howTo approach some of these questions, we ever, that constant contact with interstitial will need systematic studies to establish the cells is not required for maintenance of the localization of type II cells in developing and type II cell morphology and many other adult lungs. Current survey of the literature differentiated features of this cell type. suggests that type II cells localize at alveolar Cell-to-celladhesion molecules(CAMs and corners; however, this issue has not been cadherins) have been shown to be important rigorously evaluated. Such studies may lead for normal differentiation, migration, and tis- to identification of specific basement memsue morphogenesis (69-71). Although no spe- brane component(s) or other factors that may cificstudies have addressed this family of mol- be important for epithelial cell differentiation. ecules during lung repair and specific disease Studies are needed to evaluate the interacstates, such interactions are important for nor- tions of the pulmonary endothelium and epmal development and the maintenance of a ithelium and how such interactions may regufluid-free alveolar space. Observations from late the differentiation of the respiratory epideveloping systems suggestthat even transient thelium during development and repair. contact of one cell type with another, preBecause basement membrane influences sumably facilitated by CAMs, may determine may be mediated not only by the composithe path of differentiation for that cell type, tion but also by the three-dimensional orgaand potentially its role in subsequent repair nization of the constituents, studies are needprocesses. Cadherins are a family of related ed to compare observations made in systems cell-cell adhesion molecules (71). Hirai and using purified basement membrane compocoworkers (72) have reported that antibodies nents with those made in situ and in systems to members of this family inhibit normal using native or reconstituted basement memmouse lung morphogenesis, thus demonstrat- branes. In this respect, culture of type II cells ing a role for the cadherins in lung develop- on an intact alveolar basement membrane ment. Although such interactions during de- when combined with use of antibodies to spevelopment may define the limits within which cific basement membrane components may a type II stem cell may differentiate, their role aid in identification of the factors that reguin determining the localization of type II cells late type II cell localization at specific sites. in normal lungs or during repair remains to Finally, studies in this area will be expeditbe established. ed by the use of recently identified cell surMechanical factors are important for dif- face differentiation markers for type I (75) ferentiation in many systems. Flexibility of and II pneumocytes (76-78). the substratum has been shown to be important for morphology and function of type II Conclusion alveolar epithelial cells in vitro (44, 45, 73). In vivo, atelectasis results in decreased sur- In adult lungs, the numerical ratio of type factant secretion. In contrast, lung inflation I to type II cells remains constant, at least stimulates surfactant secretion (37, 74), and in part, because the relative areas of alveolar fluid absorption in neonatal lungs. Meehan- epithelial basement membranes that deter-



mine the two cell types also remain constant. Interaction of the epithelium with the capillary endothelium through their composite basement membrane domains may be important for establishment of a normal respiratory epithelium during development and repair. The proposed hypothesis is consistent with current understanding of pulmonary physiology. Localization of cuboidal type II cells over thin segments of alveolar septa would increase the tissue barrier through which gas diffusion occurs and impair gas exchange. However, regulation of epithelial cell differentiation by the capillary endothelium would ensure localization of type I cellsoverthe thinnest segments of the alveolar septa. Type II cell phenotype would be maintained on other areas of the basement membrane. Determination of the molecular mechanisms by which lung basement membrane regulates epithelial cell functions and differentiation should continue to provide an exciting area of investigation.

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Matrix-driven pneumocyte differentiation.

This commentary presents evidence in support of a hypothesis that adult mammalian alveolar epithelial basement membrane possesses functional and struc...
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