Postnatal Development of the Lamina Reticularis in Primate Airways

Authors

  • Michael J. Evans,

    Corresponding author
    1. California National Primate Research Center, University of California, Davis, California
    2. Center for Comparative Respiratory Biology and Medicine, University of California, Davis, California
    • VM: APC, One Shields Avenue, University of California, Davis, CA 95616
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    • Tel: (530) 754-7540. Fax: (530) 752-7690.

  • Michelle V. Fanucchi,

    1. Department of Environmental Health Sciences, School of Public Health, University of Alabama, Birmingham, Alabama
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  • Charles G. Plopper,

    1. California National Primate Research Center, University of California, Davis, California
    2. Center for Comparative Respiratory Biology and Medicine, University of California, Davis, California
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  • Dallas M. Hyde

    1. California National Primate Research Center, University of California, Davis, California
    2. Center for Comparative Respiratory Biology and Medicine, University of California, Davis, California
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Abstract

The basement membrane zone (BMZ) appears as three component layers: the lamina lucida, lamina densa, and lamina reticularis. The laminas lucida and densa are present during all stages of development. The lamina reticularis appears during postnatal development. Collagens I, III, and V form heterogeneous fibers that account for the thickness of the lamina reticularis. Additionally, there are three proteoglycans considered as integral components of the BMZ: perlecan, collagen XVIII, and bamacan. Perlecan is the predominant heparan sulfate proteoglycan in the airway BMZ. It is responsible for many of the functions attributed to the BMZ, in particular, trafficking of growth factors and cytokines between epithelial and mesenchymal cells. Growth factor binding sites on perlecan include FGF-1, FGF-2, FGF-7, FGF-10, PDGF, HGF, HB-EGF, VEGF, and TGF-β. Growth factors pass through the BMZ when moving between the epithelial and mesenchymal cell layers. They move by rapid reversible binding with sites on both the heparan sulfate chains and core protein of perlecan. In this manner, perlecan regulates movement of growth factors between tissues. Another function of the BMZ is storage and regulation of FGF-2. FGF-2 has been shown to be involved with normal growth and thickening of the BMZ. Thickening of the BMZ is a feature of airway remodeling in asthma. It may have a positive effect by protecting against airway narrowing and air trapping. Conversely, it may have a negative effect by influencing trafficking of growth factors in the epithelial mesenchymal trophic unit. However, currently the significance of BMZ thickening is not known. Anat Rec, 293:947–954, 2008. © 2008 Wiley-Liss, Inc.

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).

Figure 1.

(A) The epithelial mesenchymal trophic unit (EMTU) consists of a layer of epithelial cells (epithelial cell layer), the basement cells membrane zone (BMZ), and the attenuated fibroblast sheath (mesenchymal cell layer) (Evans et al.,1999, Am J Respir Cell Mol Biol 21:655–657). (B) High-power light microscopy of a representative portion of the EMTU, illustrating the BMZ (between arrow heads) and nuclei of cells in the epithelium and attenuated fibroblast sheath lining its mesenchymal surface (hematoxylin and eosin). Bar: 10 μm.

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.

Figure 2.

The width of the l. reticularis at different airway levels is variable. It decreases in width as the airways branch, and decrease in circumference from the trachea to the smaller airways.

Table 1. Characteristic of the basement membrane
Basement membrane (light microscopy)Basal lamina (electron microscopy)Basement membrane zone (molecular structure)
  Cellular interface
Basement membraneLamina lucidia Collagen (XVII)
   Laminin (5,6, and 10)
   Integrins (α6β4)
  Cellular-matirx interface
   Collagen (IV)
   Laminin (1)
 Lamina densa Entactin/Nidogen
   Proteoglycans (perlecans, bamacan, agrin, collagen XVIII)
   Stored growth factors (FGF_2)
  Matrix interface
 Lamina reticularis Collagen (I, III, V, VI, and VII)
   Proteoglycans (perlecan, bamacan, collagen XVIII)
   Stored growth factors (FGF-2)

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).

Figure 3.

Immunohistochemistry of collagen I (between arrowheads) illustrating how dense and distinct the lamina reticularis is when compared with the remainder of the extra cellular matrix. This illustration is in sharp contrast to the lamina reticularis stained with hematoxylin and eosin shown above in Fig. 1A. Bar: 20 μm.

Figure 4.

Fluorescent light micrograph of lamina reticularis (LR) autofluorescence in a tracheal whole mount. (A) Most of the autofluorescent fibers are large, entwined with and parallel to each other and oriented with the longitudinal axis of the airway. The layer of autofluorescent fibers lies just beneath the epithelium and is thin compared to the rest of the tracheal wall (TW) which is not autofluorescent. Bar: 80 μm. (B) Much smaller autofluorescent cross-linking fibers are against the dark tracheal wall (arrowheads). They are oriented at approximately right angles to the large fibers. Bar: 40 μm. (C) Openings (arrows) in the lamina reticularis are often observed near the cartilage rings. Bar: 80 μm (Reproduced with permission from Evans et al., Am J Respir Cell Mol Biol,2000, 22, 393–397, American Thoracic Society).

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).

Figure 5.

Diagram of the EMTU listing several molecules associated with epithelial mesenchymal interactions that have binding sites on perlecan. The position of perlecan between the epithelial and mesenchymal layers demonstrates how it could regulate extracellular trafficking of these molecules.

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.

Figure 6.

Immunohistochemistry of perlecan as an intergral component of the BMZ (arrowheads). Perlecan is also present around blood vessels (arrow). Bar: 20 μm.

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).

Figure 7.

(A) Immunohistochemistry of FGF-2 in the BMZ (arrowheads). FGF-2 is bound to the perlecan component of the BMZ. (B) FGFR-1 immunoreactivity is expressed on the surface and cytoplasm of basal cells and cilia. (C) Syndecan-4 immunoreactivity is expressed on the surface and cytoplasm of basal cells. Bar: 20 μm.

Figure 8.

Illustration of BMZ bound FGF-2, extracellular signaling via diffusion or FGF-binding protein (FGF-BP) and formation of the FGF-2 ternary complex with basal cells of airway epithelium. (Perlecan + FGF-2 ↔ 2FGF-2 + 2FGFR-1 + 2Syndecan-4 → Tyrosine Kinase Signaling) (Reproduced with permission from Evans et al., Am J Physiol Lung Cell Mol Physiol2003, 285, L931–L939, American Physiological Society).

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).

Figure 9.

Graph demonstrating postnatal growth in width of the lamina reticularis. Growth appears to be completed between 6 and 12 months of age.

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.

Figure 10.

(A) FGF-2 immunoreactivity at 1 month is associated mainly with basal cells (BC) and the lateral intercellular space (arrows). It is not apparent in the BMZ or ECM. (B) FGF-2 immunoreactivity at 6 months is now present mainly in the BMZ (arrows). Weak immunoreactivity is associated with BC and the lateral intercellular space (arrows). Bar: 20 μm.

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.

Acknowledgements

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.

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