The basement membrane is the central component of the epithelial mesenchymal trophic unit (EMTU). This anatomical unit consists of opposing layers of epithelial and mesenchymal cells separated by the basement membrane (Evans et al.,1993,1999; Holgate et al.,2000) (Fig. 1). The basement membrane has a number of functions in the EMTU. It is specialized for attachment of epithelium with the underlying extracellular matrix; serves as a barrier; binds specific growth factors, hormones, and ions; is involved with electrical charge, cell–cell and cell–matrix communication (Adachi et al.,1997; Crouch et al.,1997; Sannes and Wang,1997). An important function of the basement membrane is regulating the exchange of information between epithelial and mesenchymal tissues (Minoo and King,1994).
In the electron microscope, the basement membrane appears as three layers: the lamina lucida, the lamina densa, and the lamina reticularis. Together they form the basal lamina. The lamina reticularis is the basal portion of the basement membrane. It is also the portion that is visible with the light microscope and becomes thickened in asthma. It is often referred to as the reticular basement membrane or subepithelial basement membrane. The lamina reticularis is variable in its distribution anatomically and in its thickness. It is not apparent in all tissues; however, it is well developed under multilayered epithelium. The lamina reticularis is especially pronounced under the respiratory epithelium of the trachea, where it may be up to 20.0 μm thick. It becomes thinner as it extends from the trachea into the small airways and alveoli (Fig 2). Structurally, the lamina reticularis functions as a region of attachment between the lamina densa and the extracellular matrix (Yurchenco and O'Rear,1994; Adachi et al.,1997; Sannes and Wang,1997; Erickson and Couchman,2000). Functionally, it acts as a gate keeper by regulating the movement of cytokines, chemokines, and growth factors between epithelial and mesenchymal tissues. When studying the molecular structure and function of the basal lamina, it is commonly referred to as the basement membrane zone (BMZ). The structure and molecular composition of the BMZ are given in Table 1.
The lamina reticularis consists of numerous collagen fibrils. Immunohistochemical studies have shown that the collagen fibrils consist primarily of types I, III, and V collagen (Evans et al.,2002a) (Fig. 3). Collagen types I, III, and V form heterogeneous fibers that account for the thickness of the lamina reticularis (Evans et al.,2002a). These fibers are not randomly arranged, but instead appear as a mat of fibers oriented along the longitudinal axis of the airway. Smaller fibers are crosslinked with the larger fibers to complete this structure (Evans et al.,2000) (Fig. 4). These collagen fibers form the structural framework of the lamina reticularis. They are thinner than other fibers in the extracellular matrix (ECM) and have fewer bands indicating that the lamina reticularis is distinct from the rest of the ECM (Saglani et al.,2006). Anchoring fibrils of type VII collagen loop through strands of collagen fibers in the lamina reticularis and then reattach to the lamina densa (Nievers et al.,1999). In this way, the epithelium is attached to the underlying extracellular matrix. The lamina reticularis is thought to be attached to the ECM with oxytalan of the elastic fiber system (Bock and Stockinger,1984; Leick-Maldonado et al.,1997; Mauad et al.,1999; Evans et al.,2000).
Proteoglycans are the other main structural component of the BMZ. There are three proteoglycans that are considered to be an integral component of the BMZ in the airways: perlecan, collagen XVIII, and bamacan. These proteoglycans are found in basement membranes throughout the body and are specifically classified as BMZ proteoglycans (Halfter et al.,1998; Iozzo,1998). Their spatial localization in the BMZ implies specific functions for these proteoglycans. The large number of molecular binding sites present on these proteoglycans suggests that one of their functions would be related to trafficking of specific molecules within the EMTU (Fig. 5).
Perlecan is considered to be the predominant heparan sulfate proteoglycan in the airway BMZ and has been studied more than the other two proteoglycans (Fig. 6). It is responsible for many of the functions attributed to the BMZ, in particular, the trafficking of growth factors and cytokines between epithelial and mesenchymal cells (Iozzo,1998). Growth factor binding sites on perlecan include FGF-1, FGF-2, FGF-7, PDGF, HGF, HB-EGF, VEGF, and TGF-β (Segev et al.,2004). Growth factors pass through the BMZ when moving between the epithelial and mesenchymal cell layers. They move by rapid reversible binding with sites on the heparan sulfate chains and core protein of perlecan (Dowd et al.,1999). In this manner, perlecan can regulate movement of growth factors between tissues (Iozzo,1998,2001). When released from perlecan, growth factors can initiate cell proliferation, the production of other growth factors and cytokines, cell surface receptors and molecules such as collagen and other proteoglycans.
A specific function of perlecan is the storage and regulation of FGF-2. FGF-2 is a ubiquitous multifunctional growth factor that is stored in the BMZ of most tissues and organs (Iozzo,1998). It is stored in the BMZ by binding with perlecan. When bound to perlecan, FGF-2 is inactive and also protected from proteases. FGF-2 can be released from perlecan in response to various conditions and become an extracellular signaling molecule (Dowd et al.,1999; Shute et al.,2004). FGF-2 released from the BMZ forms ternary signaling complexes with FGFR-1 and syndecan-4 on target cells (Fig. 7). In large airways, the target cells are basal cells and in small airways Clara cells (Evans et al.,2003) (Fig. 8). The significance of BMZ-associated FGF-2 signaling in airway epithelium has not been determined. It is known to play important roles during development and as a regulator of growth and differentiation in the adult (Bikfalvi et al.,1997). In the lung, FGF-2 may be associated with regulation of a number of molecules associated with growth and repair of the airway, e.g., FGFs, epidermal growth factor, endothelin-1, and TGF-β (Holgate et al.,2000).
DEVELOPMENT OF THE LAMINA RETICULARIS
The lamina reticularis develops postnatally in primates during the first 6 months of life (Evans et al.,2002b) (Fig. 9). Collagen I is not expressed in BMZ during fetal lung development. Collagen III expression is light and discontinuous in the epithelial BMZ (Wright et al.,1999). Although collagens I and III are not expressed during the early stages of fetal development, collagen V is expressed in the early stages (Wright et al.,1999). Collagen V is associated with determining the diameter of collagen fibrils and its early appearance indicates an important role in fiber formation. Postnatal growth is characterized by a patchy pattern of thick and thin areas of collagen fibers (Evans et al.,2003,2004). With continued growth, the thin areas decrease and there is an increase in the average width of the reticular BMZ. Proteoglycans are associated with the collagen fibers during all phases of development of the lamina reticularis. These studies indicate that normal growth of the BMZ is not uniform throughout the BMZ but occurs as foci of synthetic activity (allometric growth).
Studies show that development of the lamina reticularis is associated with ternary signaling of FGF-2 through basal cells (Evans et al.,2002a). The receptors for FGF-2 ternary signaling, FGFR-1 and syndecan-4, are expressed by basal cells at all time points during lung/airway development. During the first 3 months of development, FGF-2 is strongly expressed in basal cells. However, by 6 months of age, FGF-2 is expressed primarily in the lamina reticularis and only weakly in the basal cells (Fig. 10). This corresponds with a decrease in growth of the lamina reticularis in width observed between 6 and 12 months of age (Fig. 9). The identities of signaling molecules released by epithelial basal cells treated with FGF-2 have not been determined directly. However, a number of studies have shown that signals from the epithelium stimulate the underlying attenuated fibroblast/myofibroblast sheath to synthesize BMZ collagen. Presumably, the signaling molecules released by basal cells stimulate the underlying fibroblast/myofibroblast layer to synthesize the collagen of the BMZ.
Extracellular signaling molecules are regulated in part through binding with perlecan as they move through the BMZ to receptors on the fibroblast/myofibroblast layer of cells. A recent study illustrated the importance of perlecan in the developing BMZ (Evans et al.,2003). It was shown that exposure to ozone depleted the BMZ of perlecan and there was atypical development of the BMZ. FGF-2 immunoreactivity was present in basal cells, the lateral intercellular space, and attenuated fibroblasts, but not in the BMZ. The cell surface proteoglycan, syndecan-4, was upregulated in the basal cells, suggesting it had taken the place of perlecan in the regulation of FGF-2. Thus, in the absence of perlecan, alterations in regulation of FGF-2, FGFR-1, and syndecan-4 (and presumably other growth factors) were associated with abnormal development of the BMZ. This study was performed in primates and is directly relevant to human disease.
THICKENING OF THE LAMINA RETICULARIS IN LUNG DISEASE
Thickening of the lamina reticularis is a characteristic feature of airway remodeling in the lungs of asthmatics (Bousquet et al.,2000). However, it is also not unique to asthma. Thickening of the lamina reticularis has also been reported in eosinophil bronchitis (Milanese et al.,2001; Brightling et al.,2003), lung transplant recipients (Ward et al.,2002), allergic rhinitis (Bousquet et al.,2004), and chronic obstructive lung disease (Kranenburg et al.,2006). Thickening occurs early during the development of asthma in symptomatic children 1 year and older (Cokugras et al.,2001; Payne et al.,2003; Pohunek et al.,2005). Increases in the thickness of the lamina reticularis are correlated with other remodeling changes in the airway, such as increases in smooth muscle, submucosal glands, and inner wall area (Cokugras et al.,2001; James et al.,2002; Kasahara et al.,2002). However, the amount of thickening is not correlated with the severity of the disease (Chu et al.,1998; Benayoun et al.,2003). It is not clear how widespread thickening of the lamina reticularis is throughout the lung; however, this condition has been reported in the upper and lower respiratory tract in asthmatics (Jeffery,2001) and in experimental models of asthma (Schelegle et al.,2001; Evans et al.,2002b). In addition, lamina reticularis thickening has been reported in the lungs of children before the onset of asthma (Bush,2008). This information suggests that thickening of the lamina reticularis is a general characteristic that occurs throughout the airways and is an intrinsic part of the asthma phenotype. The process of lamina reticularis thickening in asthma is probably the same as that observed in normal development, i.e., signals from the basal cells stimulate the underlying attenuated fibroblast sheath to synthesize and assemble components of the lamina reticularis (Evans et al.,2002a).
The significance of lamina reticularis thickening in asthma is not clear. There are several possible effects that thickening may have on the lung. It may have a positive effect by physically protecting against airway narrowing and air trapping (Milanese et al.,2001). Thickening may also increase the proteoglycan content and increase the capacity of the lamina reticularis to process trafficking cytokines and growth factors in the EMTU. However, thickening of the lamina reticularis could also decrease this process and affect various functions in the epithelial-mesenchymal trophic unit in a negative way (Davies and Holgate,2002). It has been suggested that thickening may be associated with abnormalities in the epithelium concerning sloughing, repair, and mucous cell hyperplasia (Holgate et al.,2000; Polosukhin et al.,2007). Additionally, it was shown that a thickened lamina reticularis could change the pattern of the airway folding resulting in increased airflow obstruction (Wiggs et al.,1997). This concept is strengthened by the fact that even slight increases in thickness can affect respiratory function (Shiba et al.,2002). However, currently it has not been shown clearly what effects thickening of the lamina reticularis has on functions of the airways.
The authors thank Susan Nishio and Melinda Carlson for preparation of the figures and editing of the manuscript. They acknowledge the staff at the Respiratory Diseases Unit at California National Primate Research Center for their technical assistance, and the members of the Comparative Respiratory Biology Group at UC Davis for their collaborative efforts in this study.