Structural and functional relationships between type II pneumocytes and components of extracellular matrices.

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Mini-Review

Structural and Functional Relationships Between Type I1 Pneumocytes and Components of Extracellular Matrices Philip L. Sannes

ABSTRACT: Type 11pneumocytes of the pulmonary alveolus are dynamic cells with multiple functional capabilities in vivo, including secretion of su$ace-active lipoproteins and cell renewal of the epithelial lining of the alveolus, involving its differentiation into another cell type (the type I pneumocyte). The factors that injluence and control these processes, which are vital to the function of the alveolus, have begun to be more clearly understood in recent years, in large part because of the development of adequate in vitro systems, which permit the manipulation of relevant variables. a e s e appear to be a complex interaction between insoluble components of extracellular matrices, principally of the basement membrane, and soluble factors that include hormones and growth factors. This review focuses particularly on those components of extracellular matrices that spectfically and nonspecifically impact on t p e II cell function, and it attempts to bring together the diverse technical approaches used to define and examine these relationships cytochemically and functionally.

INTRODUCTION The pulmonary alveolus is comprised principally of two major cell types: the type I and type I1 pneumocytes. The type I cell is morphologically a large but attenuated, squamous type of cell and covers 90% to 95% of the alveolar surface area [l, 21. It constitutes 5% to 8% of the total lung cell population and serves as a cellular barrier between the pulmonary parenchyma (connective tissue and blood vessels) and the alveolar space. The type

From the Department of Anatomy, Physiological Sciences, and Radiology, College of Eterinary Medicine, North Carolina State University, Raleigh, North Carolina. Address all correspondence to Philip L. Sannes, Ph.D., APR-NCSU Veterinary College, 4700 Hillsborough Street, Raleigh, NC 27606. Received 23 August 1990; accepted 28 August 1990.

Experimental Lung Research 17:639-659 (1991) Copyright 0 1991 by Hemisphere Publishing Corporation

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I1 cell is a smaller, more cuboidal cell, roughly double in number to the type I cell, and has long been recognized for its production and secretion of surface-active material [3]. More recently, however, it was shown to possess additional (if not varied) functional capabilities, including xenobiotic metabolism [4, 51 and transepithelial movement of water [6]. Perhaps most significant were the observations of Evans et al. [7] and Adamson and Bowden [S], who demonstrated in whole animals that these cells were capable of dividing and who proposed that they represented the progenitor for cells of the alveolus. This assertion was reinforced when it was proved that type I1 cells were responsible for reepithelialization of the alveolus following alveolar damage [9, lo]. This process was shown t o have particular significance in repair because its interruption or delay resulted in a pathogenetic outcome [11-131. Therefore, type I1 cells are not only responsible for maintaining stable cell populations in the normal lung, but they are critical determinants of cell regeneration following injury. Techniques that isolate pure populations of type I1 cells have permitted more in-depth study of this important cell and its multifaceted role in the alveolus. It is now appreciated that type I1 cells in culture are not only very sensitive and responsive to components of connective tissue matrices (generally referred to as extracellular matrices [ECM]) but also are capable of synthesizing them as well [ 14-16]. Generally, the many components of ECM, acting singly and in combinations, are known to affect such basic functions of cells as migration, proliferation, and differentiation [ 171. The type I1 cell’s responsiveness and capacity to “condition” its extracellular environment make it an even more compelling central figure in the normal and pathogenetic processes characteristic of the pulmonary alveolus. It is the purpose of this review to examine what is known about the structural and functional relationships between type I1 cells and extracellular matrices, interpret them in light of the rapidly expanding data base, and suggest some possible future directions for this relatively new and exciting area of pulmonary biology. To accomplish this, alveolar basement membranes and their cytochemical composition are first examined to define the rationale for studying specific ECM-cell interactions. Next, the criteria used for evaluating these interactions in vitro-specifically, the structural and functional characteristics of type I1 cells-are considered. ALVEOLAR BASEMENT MEMBRANES: SPECIALIZED EXTRACELLULAR MATRICES The basement membranes of the alveolar region represent the primary ECMs, which interface with the alveolar epithelium [18, 191. In the broadest terms, basement membranes are generally divided into three main regions: a lamina lucida, immediately adjacent t o the overlying epithelium, a lamina


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densa, which is subjacent to the lamina lucida and is usually electron dense in specimens routinely prepared for transmission electron microscopy, and a lamina reticdaris (or fibroreticularis), which connects the lamina densa to the underlying connective tissue stroma [20, 211. The term basal lamina is the classic histologic term for basement membrane and is often used interchangeably with lamina densa. This terminology is not used here, rather the more inclusive term basement membrane (all three layers) is employed exclusively. The basement membrane of the pulmonary alveolus is particularly unique in regions where the alveolar epithelium is closely aligned with the endothelium of the pulmonary vasculature. Here, the basement membranes of the respective epithelia are shared in most species studied. This results from a fusion of the two basement membranes, which occurs during late pulmonary development [22]. In these fused regions there is no lamina reticularis because there are no underlying parenchymal elements. The lamina lucidi of the respective epithelia, now separated only by a common lamina densa, are called lamina rarae. That lamina rara associated with the alveolar side is referred to as the lamina ram externa (the alveolus is “external” to organ parenchyma), whereas its counterpart of the endothelial side is the lamina rara interna. Cytochemically Definable Anionic Sites in the Alveolar Basement Membrane Until recently, because little was known about their composition, the basic structural constituents discussed above represented the complete state of our knowledge concerning pulmonary basement membranes. Then, Katsuyama and Spicer [23] investigated this structure cytochemically using cationic dyes with reaction products that provided sufficient contrast for the structure to be seen with the aid of the transmission electron microscope. Dialyzed iron [24] and high-iron diamine [25], which react with anionic end groups of carbohydrates and glycosaminoglycans, were employed to demonstrate for the first time that the alveolar basement membrane in the rat was asymmetrical in composition, relative to alveolar and capillary sides [23]. Reactive sites were concentrated within the lamina rara externa, which the researchers suggested may represent a kind of “cation-retaining’’ layer between the alveolus and blood vasculature. Shortly thereafter, Vaccaro and Brody [26], using a different cationic dye, ruthenium red, showed a similar asymmetry in the rat alveolar basement membrane. This dye was no more specific than those used in the Katsuyama and Spicer [23] study, but its reaction product had the advantage of being granular, or punctuate, in nature. This enabled them to not only count particles, but to assess the regularity (pattern) of their appearance within ultrastructural profiles of alveolar basement membranes.


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Accordingly, they found that 79% of the ruthenium red-reactive sites were found in the lamina rara externa and that they were 55 to 65 nm apart 1271. The reactive sites did not change relative to shared or unshared regions of the alveolar basement membrane. The relationship of basement membrane regions to different cell types was not analyzed; because cell-lysing detergents were used in the cytochemical protocols t o enhance reagent penetration, distinguishing morphologic characteristics were lost. The researchers were, however, able to partially characterize the reactive sites by digesting specimens with specific glycosidases before cytochemical treatment. In this way, they showed that those found in the lamina rara externa of the alveolar basement membrane probably reflected the presence of heparan sulfate, whereas those found in the unshared endothelial basement membrane were heparan sulfate plus other sulfated glycosaminoglycans that could not be characterized [27]. Brody et al. [22] reported that during early postnatal development in the rat, there was twice as much ruthenium red-reactive heparan sulfate in the alveolar basement membrane at birth than 8 days later and five times that found in the adult. In addition, they showed that, in the alveolar basement membrane, ruthenium red-reactive anionic sites were symmetrically disposed until about 8 days after birth, at which time the typical asymmetry described above was detectable. Using the same technique, Grant et al. [28] observed that the distribution of ruthenium red-reactive anionic sites within the basement membrane appeared to differ between type I and type I1 cells during fetal growth in the rat. They showed that the size and density of ruthenium red staining of the basement membrane was less beneath type I1 cells than beneath type I cells. The study of these anionic sites was further probed with the more sulfatespecific high-iron diamine (HID) method [25, 29j using a technique that intensifies the reaction product at the electron microscopic level [30].This approach proved quantifiable and demonstrated that the basement membrane microdomains of the type I and I1 pneumocytes were significantly different in the adult rat and rabbit [31]. The lamina rara externa associated with type I cells had more than twice as many HID-reactive anionic sites as the same region associated with type I1 cells, whether or not its basement membrane was shared (Fig. 1). The asymmetry of reactive sites seen previously [23] was confirmed quantitatively, but the anionic sites detected with HID were distributed nearly symmetrically with respect to the three layers in the basement membrane associated with the type I1 cell. Van Kuppevelt et al. showed very similar results in mouse [32, 331 and human [34] alveolar basement membranes using cuprolinic blue-yet another electron-dense cationic dye that reacts with similar anionic components of ECM as HID and ruthenium red. The reaction product with this procedure, however, tends to be filamentous in nature, possibly reflecting the nature (perhaps, the charge density) of the reactive molecules, which are


Figure 1 Representative electron micrographs of rat lung fixed 1 h in 1% paraformaldehyde-1% glutaraldehyde in 0.1 mol/L cacodylate-HCl buffer and treated with high iron diamine method for localization of sulfated complex carbohydrates [25]. The reaction product has been intensified with silver proteinate [30], which accounts for the round silver grains (mil arrowbed). (a) The basement membrane that is shared by a type I pneumocyte (0 and adjacent endothelial cell (4 is delineated between the two large arrows. The lamina rara externa, which lies immediately adjacent to the type I cell, is “preferentially” labeled with silver grains (small arrowheads), resulting in an asymmetric pattern of reactivity. A represents the alveolar space ( x 100,000, bar 0.1 pmeter). (b)Electron micrograph sample from an adjacent alveolar region treated as above, showing the basement membrane between adjacent large arrows, shared by a type I1 pneumocyte (14 and neighboring endothelial cell (4.The basement membrane appears symmetricully reactive with respect to the distribution of silver grains. The type I1 cell contains a typical multivesicular body (MV), which aids in its identification ( x 100,000; bar 0.1 pmeter).

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presumed to be glycosaminoglycans associated with basement membrane proteoglycans. The differences in reaction-product distribution they observed was reflected in the noticably longer (on average) filaments associated with type I cell basement membranes as compared with those associated with the same region of the type I1 cell. The reactive sites were characterized as chiefly representing heparan sulfate-containing proteoglycans [33]. Despite the somewhat diverse technical approaches used in these cytochemical studies, they provide consistent and compelling evidence that the alveolar basement membrane is something of a “mosaic” of anionic sites. These sites are concentrated asymmetrically on the alveolar side of basement membrane areas occupied by type I cells (hence, >90% of the alveolar basement membrane surface area). In regions occupied by type II cells, the number of anionic sites is greatly diminished. This unique asymmetry is present regardless of whether or not the alveolar basement membrane is shared with the vascular endothelium. The vast majority of reactive sites, presumably reflecting the molecular arrangement of charged anionic end groups of proteoglycans, are found in the lamina rara externa or lamina lucida regions, whereas the other basement membrane regions of the alveolus are significantly less reactive. It is also clear from these studies that the basement membrane microdomains associated with type I cells are distinctly different from those of type I1 cells. These differences appear to be quantitative, because the degree of reactivity is significantly greater beneath the type I cell. Enzymatic digestion procedures suggest that the cytochemically reactive sites in the alveolar basement membrane are principally heparan sulfate, probably in the form of one or more complex proteoglycans.

Immunocytochemically Definable Sites in the Alveolar Basement Membrane Immunocytochemical examination of alveolar basement membranes has helped complete our current understanding of their structure and composition and place it in perspective with basement membranes found in other organ systems. Madri and Furthmayr [35] were the first to immunohistochemically describe type IV collagen in pulmonary basement membranes. This unique collagen is found exclusively in basement membranes and provides them with a strong but flexible structural scaffolding [36]. It also has important adhesive properties for cells and attachment domains for other ECM components [37]. Sano et al. [38], Amenta et al. [39], and Coulombe and Bendayan [40]have confirmed its presence in the lung and its confinement to basement membranes by immunostaining for transmission electron microscopy. Its location within the basement membrane is generally thought to be within the lamina densa [21, 411, although this is difficult to resolve precisely with most immunostaining protocols. Laminin, a unique basement membrane glycoprotein, has also been localized ultrastructurally


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in the alveolar basement membrane [42]. It possesses strong affinity for type


IV collagen [41] and, in other organs, is found chiefly in the lamina densa. It has been suggested that laminin may also extend into the adjacent laminae [21]. Fibronectin is often regarded as a component of many basement membranes, and it has been immunolocalized in alveolar basement membrane at the level of the electron microscope [42, 431. However, its cytochemical localization there could be interpreted as the result of preparation artifact following fixation of plasma fibronectin within basement membranes during vascular perfusion [42]. This conclusion is due to its somewhat “spotty” reactivity. Fibronectin was presumed to be forced out of the plasma and enmeshed and fixed within the alveolar basement membrane; in this way, it was detected with immunostaining procedures. Regardless of its origin, fibronectin’s potential role in basement membrane-cell interactions in the lung must be considered. Another basement membrane component, heparan sulfate proteoglycan, has also been reported to be present in rat alveolar basement membrane and to be detectable by immunostaining at light and electron microscope levels [44]. As indicated above, none of these immunostaining procedures has permitted sufficient resolution to demonstrate either an asymmetrical distribution of reaction product or differences between type I and type I1 cells. This is not surprising, because all but one of the existing reports on immunolocalization of basement membrane components in the alveolus employed peroxidase as a detecting molecule. Peroxidase-reaction product is inherently flocculent with the tendency to diffuse-thus impeding an accurate definition of the microanatomic location of antigenic sites [45]. In the one study using colloidal gold for detection [40], none of the differences related to asymmetry and cell type demonstrated with cationic dyes were reported. Considering that immunostaining and cationic dye procedures localize either completely different molecular components of the same macromolecule or different molecules altogether, this lack of corroboration might be predictable. This is well illustrated by what is known about proteoglycans [46], which are composed of heavily anionically charged glycans that would be expected to react with cationic dyes, whereas their protein cores would tend to be more likely targets of appropriately directed antibodies. Taking into account their considerable size, reactive site locations for the same molecule might be found in different regions of the same basement membrane microdomain. The lack of agreement between studies using immunoreactivity and cationic dye-reactivity should not diminish the importance of the data, which strongly point to asymmetry and cellassociated differences within the alveolar basement membrane. The sulfated glycoprotein entactin, also a known component of basement membranes, might be expected to contribute to the charge density and distribution of these anatomic microdomains, but it has only been described in the lung in tissue surveys [47]. Chondroitin sulfdte proteoglycan was recently

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demonstrated to be a component of most basement membranes [48] and has been immunohistochemically localized in the basement membranes of pulmonary airways and alveoli [49-511. More in-depth study is in progress to attempt to define its ultrastructural localization to determine its possible contribution to the unique asymmetry and cell-related differences discussed above.

TYPE I1 CELL CHARACTERISTICS AS INDICATORS OF FUNCTION The above cytochemical studies provide interesting insights into in vivo cellmatrix relationships in the pulmonary alveolus. But how might the questions they raise be tested and interpreted in more functional terms? Because addressing this problem in the intact organ is nearly impossible, it is highly desirable and appropriate to use in vitro approaches. Pure populations of isolated type I1 cells and organotypic cultures derived from fetal lungs have been the primary methods of choice. Because cell-ECM interactions have not been studied with the latter preparations, the remainder of this discussion focuses on type I1 cells enzymatically isolated from adult lungs. Developing an adequate procedure for obtaining pure populations for culture was not an easy task, but it was successfully accomplished by Kikkawa and Yoneda [52]. This original method has since been refined by a number of different researchers, each with variations. Isolated cells have been used to examine a wide range of type I1 cell structural and functional characteristics. In recent years interest has increased in the importance of their interaction with ECM substrata in vitro as a means of more closely approximating their in vivo environment and probing the mechanisms of this complex relationship. But before appropriate hypotheses can be made to test the effects of matrix substrata on isolated type I1 cells, it is critical to consider those characteristics that have been used as criteria for evaluating cellular behavior. The typical phenotypic characteristics that type I1 cells express in vivo have been used as a measure of how cells change during exposure to different environments after isolation. These characteristics can be separated into two basic categories: morphologic/cytochemical and functional/synt het ic.

Morphological/Cytochemical Characteristics Diglio and Kikkawa [53] observed that isolated type I1 cells lost typical type I1 cell morphologic characteristics when placed on tissue-culture plastic, such as visible lamellar bodies, and cuboidal shape after 3 to 5 days in culture. These cells then progressively assumed the “appearance” of a type I cell (lacked lamellar bodies and had a flatter or more squamous cell shape). The morphologic transition they observed has generally been viewed as an



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expression of this cell’s recognized function as the progenitor cell of the type I cell 17, 81. When exposed to mixed ECMs synthesized by “producer” cells, such as corneal endothelium, type I1 cell morphologic characteristics could be sustained for as long as 7 to 10 days [54]. This was the first evidence to suggest that ECMs affect type I1 cells’ phenotypic expression. Acellular amnionic membrane with its preexisting basement membrane was shown to retard the natural tendency of type I1 cells to differentiate into type I cells in culture [55, 561-the cells tended to retain type I1 cell characteristics for 7 to 8 days. This contrasted with the stromal side of acellular amnionic membrane and acellular pulmonary alveolar matrix preparations, on which isolated type I1 cells rapidly (within 72 h) flattened and lost lamellar bodies [57, 581. This suggested that mixed ECMs alone were not sufficient to promote growth and/or differentiation. Other mixed extracellular matrix “cocktails,” such as that produced by the Engelbreth-Holm-Swarm (EHS) tumor, produce effects similar [15] to those seen in endothelial cellconditioned ECM and acellular amnionic membranes. These studies established an important “morphologic baseline” for subsequent type I1 cellmatrix studies that followed. Cytochemical techniques have extended these morphologic data by providing an additional perspective on the physicochemical properties of the type I1 cell. These techniques are particularly useful because they often bridge the gap that so often exists between morphologic and biochemical properties of cells. To be most useful, they should demonstrate properties relatively unique to the type I1 cell to facilitate in vivo correlation. Methods used to date have principally focused on the type I1 cell’s enzyme, lipid, and structural protein complement, as well as its unique plasma membraneassociated molecules. Each is briefly discussed here. Enzymes associated with type I1 cells were some of the first type I1 cell characteristics examined cytochemically. The presence of acid phosphatase [59, 601, aryl sulfatase B, 6-glucuronidase [60], fl-N-acetylglucosaminidase [61, 621, esterase [63], lysozyme [64], dipeptidyl peptidase 11 [65], dipeptidyl peptidase I, and cathepsin B [66] has been demonstrated in type I1 cells. They have not been routinely employed as markers of behavioral changes, but recently they were shown to be altered with time in culture on plastic [62]. Certain proteases were reported to be reduced in expression relative to the in vitro conditions, including matrix substrata composition [66]. The cell surface enzyme alkaline phosphatase was recently shown to be a potentially useful phenotypic characteristic relatively unique to type 11 cells compared with other alveolar cells. First described by Sorokin [59] and Kuhn [67] in the type I1 cell, it has been used to quantitate their proliferative response to silica in vivo [68]. This approach may have some promise, although it has not been evaluated rigorously in type I1 cells in culture. Post and Smith [69] used it to confirm the characteristics of type I1 cells isolated from fetal rat lung. It might be said that all the enzyme methods for cyto-


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chemical characterization of type I1 cells in culture have been underutilized and may yet prove useful as additional criteria for evaluating alveolar cell differentiation and regulation of intracellular processes by matrix substrata. Lipid cytochemistry has proved useful in the evaluation of type I1 cells. Mason and Williams [70] adapted a fluorescent stain (phosphine 3R) for the identification of lamellar body structures by light microscopy. This method proved useful but, as with all fluorescent probes, the staining faded with time. Mason et al. [71] then developed a tannic acid and polychrome stain that revealed lamellar bodies very effectively with an acceptable degree of permanence. Using this method, Rannels et al. [15] showed that isolated type I1 cells lost lamellar bodies when cultured on plastic. They demonstrated that this effect was retarded with exposure to laminin-coated surfaces and that EHS tumor cell matrix was even more effective in enabling type I1 cells to maintain a high content of intracellular lamellar inclusions. This stain is most useful for cytochemically evaluating one of the more unique characteristics of the type I1 cell. The immunocytochemical reactivity of the protein components of surfactant-associated lipoproteins found in lamellar bodies has been an active area of investigation, but it has not been widely applied to questions related to type I1 cell characterization and cell-matrix interactions in vitro [72]. This may be due, in part, to the fact that these molecules can be stored, as well as recycled, and therefore may be poor indicators of possible metabolic changes that occur in type I1 cells in response to alterations in their matrix environment. One of the more interesting approaches to characterizing type I1 cells cytochemically to differentiate them from other alveolar cells is immunostaining for keratin species. These structural proteins, unique to the cell of origin, have been used to identify and discriminate individual cell populations in a wide range of tissues and organs. Techniques for their immunolocalization have only recently been applied to the alveolus, but the results are compelling. Woodcock-Mitchell et al. [73] first demonstrated their usefulness by positively staining what the researchers interpreted to be type I1 cells differentiating into type I cells with a monoclonal antibody (24A,) to a 46,000 MW keratin in bleomycin-treated rat lungs. They subsequently showed that this same antibody gives increased staining intensity in isolated type I1 cells as the cells lose their typical morphologic characteristics when cultured on plastic [74]. These researchers reported that this change was retarded when cells were cultured on laminin or EHS tumor-derived matrices. The presence or absence of these cell-specific structural elements are probably essential for maintaining cell shape and morphology, which may even have an important impact on gene expression and other functional characteristics in these cells [75]. Cell surface-associated molecules have been another novel and powerful way of cytochemically examining the characteristics of type I1 cells, espe-


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cially for differentiating them from other cell types. Lectins and antibodies to unique cell surface antigens have proved useful in studies that involve the discrimination between type I and type I1 cells in vitro. Dobbs et al. [76] and Weller and Karnovsky [77] demonstrated that the plant lectin Muculu pomifru (MPA) selectively bound to the surface of type I1 cells and not type I and that Ricinus communis I (RCI) stained the surface of type I cells and not type 11. These authors showed that as type I1 cells progressed in culture (either on plastic or corneal endothelial cell-derived matrix), they lost MPA reactivity and “acquired” RCI reactivity, suggesting that type I1 cells differentiated into type I. Recent reports have questioned the use of RCI as a type I cell-specific marker [78, 791 because of its apparent reactivity with type I1 cells. The technical subtleties of this approach are considerable and are not discussed here; however, Taatjes et al. [79] suggest that Erythrinu cristuguli may be more type I cell specific. Monoclonal antibodies to plasma membrane molecules of type I1 cells promise to be another way of identifying cell types and delineating differentiation. Funkhouser et al. [80] developed an antibody to a 146,000 MW protein unique to type I1 cell membranes, and they used it to isolate type I1 cells with a cell sorter and to localize and identify the cells’ appearance during lung development [81]. Post and Smith [69] used this antibody, referred to as JBR-1, to detect maturing type I1 cells in fetal rat lungs. In unpublished preliminary studies, we have used the same antibody for immunostaining isolated cells in culture and have observed that this antigen is lost with increasing time in culture, depending on the conditions. These studies offer a wide variety of technical choices for evaluating and defining cellular phenotype of type I1 cells in culture and in vivo. Each possesses certain advantages and disadvantages. One must keep in mind that these isolated cells may have a considerable degree of phenotypic “plasticity” that is significantly influenced by environmental conditions. Therefore it is important to correlate in vitro phenotypic characteristics with those found under in vivo conditions.

Functional/Synthetic Characteristics As already mentioned, type I1 cells are now well recognized for their ability to divide and synthesize the complex components of pulmonary surfactant. These important functional, in vivo characteristics, along with the morphologic and cytochemical characteristics, have been useful criteria for probing type I1 cell functional activity in vitro. Cellular division (proliferation) in isolated type I1 cells has been studied primarily using [H’] thymidine [82]. Incorporation of [H3]thymidine into DNA can be used as an expression of DNA synthesis, and when combined with total DNA and/or cell number, a labeling index can be generated as an accurate measure of cell proliferation. Isolated type I1 cells cultured on plas-


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tic typically have a very low labeling index (0.8%) within the first 72 hours in culture, whereas those on ECM derived from corneal endothelial cells have a much higher one (18.5%)[83]. Rannels et al. [84] showed that, over 24- to 72-h period in culture, fibronectin stimulated [H3]thymidine incorporation in type I1 cells about two and one half times that of controls. They found similar increases on cells cultured on preexisting matrix substrata produced by type I1 cells and discovered that their rate of incorporation was accelerated during the 24- to 48-h period, then leveled off, whereas those on plastic had a slower, steadily rising rate over the entire period. On the other hand, laminin transiently depressed by 40% DNA synthesis using the same measure when compared with untreated plastic surfaces during the 24- to 48h period [15]. This effect was lost during the 48- to 72-h period, when laminin-exposed cells “caught up” with controls. Matrix substrata derived from the EHS tumor similarly depressed DNA synthesis during 24 to 49 h but kept levels depressed at 48 to 72 h. These effects were shown to be partially blunted by culturing cells in the presence of rat serum instead of fetal bovine serum [84]. The potential importance of this observation serves to emphasize the interplay of soluble factors, such as those in serum, and components of ECMs. Related reports were first made by Leslie et al. [82] and subsequently by Cott et al. [56] and Rannels et al. [15]. Leslie et al. [83] then showed that soluble factors derived from macrophages caused a concentration-dependent increase in type I1 cell-labeling indices, which was amplified when they were cultured on an endothelial cell-derived matrix. These interesting studies support the notion that the interaction of soluble factors and insoluble ECM components has a significant impact on type I1 cell proliferative behavior. It should be noted that, when using such indices as a measure of type I1 cell proliferation, these cells tend to synthesize DNA (incorporate labeled precursors), but not divide [83]. Clement et al. [85] recently reported that nonreplicating type I1 cells incorporate thymidine into DNA that does not appear to be in a stable form suitable for replication. They concluded from their studies that thymidine incorporation cannot be used as an indicator of type I1 cell proliferation. This suggests that, in some earlier studies, actual proliferative events (increases in cell number) may have been overestimated. As a possible alternative, Leslie et al. [83] have used autoradiography as a means of validating and establishing their “labeling index.” In this way, cytokinetic/karyokinetic events can be more accurately monitored and quantified. Recently we used 5-bromo-2 ’-desoxy-uridine (BrdU) as an alternative precursor to thymidine [51], then immunolocalized it cytochemically with monoclonal antibodies directed against it (Boehringer Mannheim, Indianapolis, IN). Like the autoradiographic approach, this enables evaluation of the relevant cytokinetic events, but without the use of isotopes. The caveat remains, however, that precursor incorporation is insufficient by itself as a measure of type 11 cell proliferation.



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Probably the most characteristic and important function of type I1 cells is their ability t o synthesize a unique profile of phospholipids, which are major components of the surface active material of the alveolus, or surfactant. The synthetic process has been evaluated by the cell's capacity to incorporate labeled precursors, such as [3H]acetate or ["C] glycerol, into phospholipids [86]. For type I1 cells, the key phospholipid products are phosphotidylcholine (PC) and phosphotidylglycerol (PG), which comprise about 65% and 9%, respectively, of the total phospholipid synthesized. These are critical indicators of whether or not type I1 cells in vitro are metabolically behaving as they are known to in vivo. Geppert et al. [87] were the first to compare surfactant phospholipid synthesis in isolated type I1 cells exposed to plastic or collagen substrata. They found that, although cells appeared to retain their typical morphologic characteristics a bit longer on rat tail collagen (mostly type I collagen), there was a progressive decline in the incorporation of ["C] glycerol into surfactant phospholipids. Lwebuga-Mukasa et al. [57] similarly showed that isolated type 11 cells plated on plastic rapidly lost their ability to synthesize their characteristic pattern of phospholipids within 72 h. However, if they were exposed to acellular amnionic membrane, this loss was modestly reduced. Dobbs et al. [76], using both ["C] acetate as a precursor and total phosphorous in phospholipid as measures, compared the effects of plastic versus matrix substrata derived from corneal endothelial cells on type I1 cell phospholipid synthesis and found that PC fell from 73% of total phospholipid at day 1 in culture to less than 50% by day 7. Similarly, PG dropped from 7% at day 1 to less than 0.5% on day 7. Lwebuga-Mukasa et al. [58] showed that, when cells were cultured on acellular matrices derived from human lung alveoli, PC levels remained unchanged at 72 h, whereas PG decreased 64% when compared at 24 h. Although later time periods were not evaluated in this study, the maintenance of PC synthesis was of considerable interest and strongly implicates basement membrane influences on the synthetic activity of these cells. Shannon et al. [88] made similar measurements on type I1 cells plated on EHS tumor-basement membrane and found that they incorporated a higher percentage of labeled acetate into PC than cells on plastic for 4 days. PG synthesis, however, was no different on matrix than on plastic. Cott et al. [56] also tested the matrix effects of acellular amnionic membrane and found that cells incorporated more labeled acetate into PC and PG than on plastic. Furthermore, they found that the presence of rat serum in place of the usual fetal bovine serum significantly elevated the levels of incorporation into PG and PC. They tested a number of different species' sera and found fetal bovine serum to be associated with the lowest combined totals for PC and PG, whereas rat serum gave the highest. This was the first study to demonstrate the potential significance of the interplay between matrix and serum factors in type I1 cell function.


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These observations were further amplified in a recent report by Kawada et al. [89], in which isolated type I1 cells were cultured on EHS tumor matrix in serum-free, hormonally defined medium. They showed that these cells incorporated increased amounts of acetate into total lipids, a higher percentage of which was incorporated into PC. The pattern of differentiated phospholipid biosynthesis they observed seems to indicate that these conditions potentiate a cellular behavior more closely similar to that seen in vivo. It might be further concluded that extracellular matrices may be a necessary condition for optimal levels of phospholipid production in type I1 cells. In a study related to the preceding discussion, Shannon et al. [75] evaluated surfactant-associated proteins (apoproteins) in isolated type I1 cells exposed to plastic, EHS tumor extract, or laminin at intervals over an 8-day period. They probed the mRNAs for selected apoproteins and found that cells cultured on plastic were uniformly negative for SP-A, SP-Byand SP-C, whereas those on EHS extract were positive for all three. Interestingly, the message for SP-A and SP-B increased with time, whereas SP-C decreased, strongly suggesting that the latter is regulated separately. Cells exposed to laminin alone were no different than those on plastic with respect to message content for these apoproteins. These compelling data, like those on phospholipid synthesis, demonstrate that an important link exists between extracellular matrix composition and cellular functions.

CONCLUSIONS AND FUTURE DIRECTIONS The studies discussed in this review represent a wide variety of technical approaches to questions related to type I1 cell behavior. The functional data strongly support the notion that extracellular matrix components, singly and in combination, play an important role in influencing type I1 cell activity. Cytochemical studies have provided important insights into the specific composition of basement membrane substrata and have even revealed novel differences in sulfation between those regions associated with alveolar type I and I1 cells. It is interesting that these differences have been attributed chiefly to the presence of heparan sulfate, which is known to have direct inhibitory effects on cell proliferation [90, 911. Although this has not been reported for epithelial cells, it may be appropriate to speculate that the high degree of cytochemically detectable sulfated components associated with the basement membrane of type I cells represent ECMs that “inhibit” their growth, whereas the less sulfated regions beneath type I1 cells are less growth inhibitory. Alternatively, it is recognized that heparin-like molecules bind and protect certain soluble growth promoters, such as basic fibroblast growth factor [92], which has been immunolocalized in basement membranes, including those of the lung [93]. Basic fibroblast growth factor can be enzymatically released from such sites [94], and in its unbound form would be expected to



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stimulate DNA synthesis [92] and possibly subsequent cellular proliferation of type I1 cells. Such a compelling scenario may be operative during certain states of development or injury and may significantly influence cell functional activity. Differences in basement membrane composition therefore may imply the differential distribution of and/or response to specific growth factors, which in turn could influence the type I1 cell’s important functional roles in normal and pathologic states. However, much remains to be understood about the complex relationships between specific matrix components and the various growth factors to which the type I1 cell is responsive. It is apparent from the above discussion that basement membrane components, such as laminin, type IV collagen, and fibronectin, are established as having effects on the activity states of isolated type I1 cells. Their effects in combination have been examined in vitro using mixtures produced by tumor cells, which has been presumed to mimic in vivo conditions. This assertion, however, is based on the assumption that all basement membranes are similar in their composition, which does not necessarily agree with known cytochemical observations indicating that basement membranes may, in fact, be heterogeneous. It seems more likely that subtle differences in specific basement membrane components and/or their combinations uniquely bind or otherwise occupy membrane receptors of the overlying cells. These receptors therefore could be modulated by such specific and nonspecific interactions with ECM elements or by intracellular structural proteins to which they are probably related on the cytosolic side of the membrane. Such mechanisms would be compatible with the multiple levels of modulation that might be predicted of a complex biologic “microsystem” as the pulmonary alveolus. It remains for future investigations to more clearly define the specifics of these complex relationships. Such studies will rely heavily on new and novel approaches, such as the recent report of the use of serum-free, defined media, to study type I1 cells in culture [89], which will enable appropriate control of the numerous variables involved and their biochemical/functional examination. Only then can the ultimate answers to these multilayered questions be gained. The author extends his gratitude to Jyotsna Khosla for the preparation of the micrographs and to Dr. Kenneth B. Adler for helpful discussions. This effort was supported in part by a grant from the College of Veterinary Medicine, North Carolina State University, and the Life Sciences Division of 3M Corporation, St. Paul, Minnesota.

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Micronodular hyperplasia of type II pneumocytes.

Protein phosphorylation and dephosphorylation in type II pneumocytes.

The relationship between lamellar bodies and lysosomes in type II pneumocytes.

Bacterial RNA polymerases: structural and functional relationships.

Genetic and structural relationships between components of a protective rhoptry antigen complex from Plasmodium falciparum.

Relationships between membrane antigens of human leukemic cells and oncogenic RNA virus structural components.

Amiloride-inhibitable Na+ conductive pathways in alveolar type II pneumocytes.

Structural and functional relationships between mouse and hamster zona pellucida glycoproteins.

Uptake and subcellular distribution of Escherichia coli lipopolysaccharide by isolated rat type II pneumocytes.

Silica exposure induces cytotoxicity and proliferative activity of type II pneumocytes.

Structural and functional relationships of human DNA polymerases.

Electron-histochemical investigations on Clara cells and type II pneumocytes of normal rat lungs.

Bioenergetic pattern of isolated type II pneumocytes in air and during hypoxia.

Structural-Functional Relationships Between Eye Orbital Imaging Biomarkers and Clinical Visual Assessments.

Epidermal growth factor transcription, translation, and signal transduction by rat type II pneumocytes in culture.

Plasminogen activator inhibitor type 1 production by rat type II pneumocytes in culture.

Process signatures in glatiramer acetate synthesis: structural and functional relationships.

Dichloroacetate Decreases Cell Health and Activates Oxidative Stress Defense Pathways in Rat Alveolar Type II Pneumocytes.

Structural and Functional insights into the catalytic mechanism of the Type II NADH:quinone oxidoreductase family.

cyclases from type II polyketide synthases.

Structural and evolutionary relationships between retroviral and eucaryotic aspartic proteinases.

Structural equation models of relationships between exercise and cognitive abilities.

Convergent and discriminant relationships between factor matrices of motor performance and arousal data.

fibrinogen matrices.