Introduction

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The epithelial basement-membrane zone is specialized for attachment of epithelium with the extracellular matrix. In addition it also serves as a barrier; binds growth factors, hormones, and ions; and is involved with cellular adhesion, electrical charge, and cell-cell communication (1). With transmission electron microscopy (TEM) the basement-membrane zone appears as three component layers: the lamina lucida, the lamina densa, and the lamina reticularis. Together, the lamina lucida and lamina densa make up the basal lamina. The lamina lucida, as seen with TEM, is a clear area between the epithelium and the lamina densa. This is thought to be a fixation artifact; however, this area actually functions as the region of attachment between the epithelium and lamina densa and contains cell adhesion molecules and anchoring filaments of laminin 5. The lamina densa is a sheet of connective tissue made up of type IV collagens, laminins, entactin, and heparin sulfate proteoglycans. On the extracellular matrix side of the lamina densa, anchoring fibrils of type VII collagen loop through strands of collagen in the lamina reticularis and then reattach to the lamina densa (4).

The third component of the basement-membrane zone, the lamina reticularis, is variable in its distribution, thickness, and composition. 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 large conducting airways, where it may be several microns thick. It becomes thicker as the airway increases in diameter. The lamina reticularis of the large airways appears as a faint magenta band in light-microscope preparations stained with periodic acid- Schiffs reagent. With TEM it can be seen that the lamina reticularis is made up of numerous collagen fibrils. Immunohistochemical studies have shown that the collagen fibrils consist of types I, III, V, VI, and VII collagen. Within and around these collagen fibrils are fibronectin, tenascin, and proteoglycans (4). Clinically, the lamina reticularis is the region of the basement-membrane zone in human large airways that accumulates collagen and leads to the subepithelial fibrosis associated with asthma (7, 8).

There is considerable information concerning the basal lamina region of the basement-membrane zone, but very little is known about the lamina reticularis. When viewed in sections with TEM the lamina reticularis is difficult to study because of its thin profile. We approached this problem by using whole mounts of airways studied by fluorescent microscopy and scanning electron microscopy. Using this approach we look down on the flat surface of the lamina reticularis rather than its thin profile. The purpose of this study was to define the structural organization of the lamina reticularis in the rat trachea. Our results demonstrate that the fibrous framework of the lamina reticularis is a complex, highly organized structure within the tracheal basement-membrane zone.

	Materials and Methods

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Animals

Tracheas from 30- to 60-d-old Sprague Dawley rats were used in this study. The methods for taking tracheal samples have been published previously (9). Briefly, the rats were killed by an overdose of pentobarbital sodium followed by exsanguination. The tracheas were removed, cut into four longitudinal sections, and placed in phosphate-buffered saline (PBS). To remove the tracheal epithelium and expose the underlying lamina densa, two methods were used. For the first method tracheal samples were placed in 50-ml Falcon centrifuge tubes containing 40 ml of 20 mM Na₂ ethylenediaminetetraacetic acid (EDTA) in Hanks' balanced salt solution minus Ca²⁺ and Mg²⁺ with 5% bovine serum albumin (BSA) at pH 7.4. The tubes were rocked for 180 min at room temperature. With a pipette, the samples were extensively rinsed with PBS to dislodge the epithelial cells (10). Other tracheal tissues were placed in 50-ml Falcon centrifuge tubes containing 0.3% of the protease Pronase E (Sigma Chemical, St. Louis, MO) with 1.0% BSA and rocked at room temperature for 180 min. The samples were then washed in PBS with a pipette to dislodge the epithelial cells as before. To remove the lamina densa and expose the lamina reticularis, Nexaband surgical adhesive (Veterinary Products Laboratories, Phoenix, AZ) was applied to the surface of the tracheal preparation with an applicator stick. After several minutes the lamina densa was removed with a rolling motion of the applicator stick, exposing the underlying lamina reticularis.

Fluorescent Light Microscopy

For microscopy, tracheal samples with the epithelium removed were fixed in 1.0% paraformaldehyde in 0.1 M phosphate buffer. The tracheal whole mounts were attached to coverslips with Nexaband surgical adhesive, placed in Petri dishes, and covered with PBS. The autofluorescence of the lamina reticularis was visualized using a Olympus blue filter (wide band) on an Olympus BH-2 fluorescent microscope and long working distance water immersion objective lenses (11).

Immunohistochemistry

Fixed airways were sliced lengthwise into equal halves. Airway halves were made permeable to antibodies by a series of dehydration/rehydration steps increasing, then decreasing, the concentrations of ethanol (70, 95, 100, 95, and 70%, 10 min each), followed by washing three times in PBS (10 min each); they were then placed in 0.3% Triton X-100 for 10 min. The tissues were then washed in two changes of PBS for 10 min each and placed in 5.0% BSA for 30 min. The tissues were placed next in a 1:100 dilution of goat antielastin (Elastin Product Co., Inc., Owensville, MO) overnight. The antibody was visualized using a biotinylated secondary antibody and streptavidin conjugated with alkaline phosphatase as supplied in the ELF kit (Molecular Probes, Inc., Eugene, OR). The substrate in the ELF kit produces a fluorescent green precipitate. The tissues were attached to coverslips with Nexaband surgical adhesive (whole mounts), placed in plastic watch glasses, and covered with PBS. The lamina reticularis was viewed with long working distance water immersion objective lenses.

Scanning Electron Microscopy

Whole mounts for scanning electron microscopy were dehydrated in a graded series of ethanol, immersed in hexamethyldisilizane, and air-dried overnight at room temperature. The samples were next coated with gold in a sputter-coater (Polaron II E5100) with 2.5 kV acceleration voltage in argon atmosphere with current of 10 mA for 2 min. Tracheal samples were imaged on a Philips scanning electron microscope 501 (Philips, Mahwah, NJ).

	Results

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Figure 1 is an example of the rat tracheal whole mounts used in this study. In tracheal whole mounts the fibrous framework of the lamina reticularis was autofluorescent and clearly visible in a fluorescent microscope. The remainder of the tracheal wall was not autofluorescent (Figure 2A). Most of the fibers were large, entwined with and parallel to each other. They were fused together in many places, appearing as a thin anastomosing mat of fibers several layers thick, oriented along the longitudinal axis of the airway. At higher magnification, the autofluorescent fiber mat was clearly visible against the dark tracheal wall. The fiber mat is made up of two populations of fibers, one larger and one smaller. The much smaller crosslinking fibers are oriented at approximate right angles to the large fibers (Figure 2B). Collectively, the fibers form a thin compact mat 1 to 2 µm thick, immediately beneath the lamina densa. In areas near cartilage rings there were often openings in the fiber mat (Figure 2C). Immunohistochemistry for elastin revealed a banding pattern on the surface of the fibers consistent with the orientation of the crosslinking fibers (Figure 3).

View larger version (64K): [in this window] [in a new window]   Figure 1.   Fluorescent light micrograph of a rat trachea whole-mount preparation (EDTA treatment). Bar: 3 mm.

View larger version (150K): [in this window] [in a new window]   Figure 2.   Fluorescent light micrograph of lamina reticularis (LR) autofluorescence in a tracheal whole mount (EDTA treatment). (A) Most of the autofluorescent fibers are large, entwined with and parallel to each other and oriented along the longitudinal axis of the airway. The layer of autofluorescent fibers lies just beneath the epithelium and is thin compared with the rest of the tracheal wall (TW), which is not autofluorescent. Bar: 80 µm. (B) Much smaller autofluorescent crosslinking fibers are visible against the dark tracheal wall (arrows). They are oriented at approximate right angles to the large fibers (pronase treatment). Bar: 40 µm. (C ) Openings (arrows) in the lamina reticularis were often observed near the cartilage rings (pronase treatment). Bar: 80 µm.

View larger version (151K): [in this window] [in a new window]   Figure 3.   Immunohistochemistry for elastin reveals a banding pattern on the surface of the fibers but not between fibers. The banding pattern is consistent with the orientation of the small crosslinking fibers. Bar: 20 µm.

The same whole mounts used for the autofluorescence studies were analyzed with the scanning electron microscope. In preparations where the epithelium was removed with protease, basal cells still covered much of the basement membrane. In some areas, columnar cells were still attached to the basal cells. Fibers were visible beneath the lamina densa (Figure 4A). In areas where the lamina densa was missing, the longitudinally oriented fibrous framework of the lamina reticularis was clearly visible. Two populations of small fibrils were oriented at approximate right angles to the large fibers. One population of these fibers was crosslinked with the larger fibers, appearing to join them together (Figure 4B). The other population of small fibers was much thinner and oriented around the surface of the large fibers (Figure 4C).

View larger version (181K): [in this window] [in a new window]   Figure 4.   Scanning electron micrograph of a tracheal whole mount after pronase treatment. (A) Basal cells (BC) remain over much of the lamina densa. In some areas columnar cells (CC) are still attached. Just beneath the lamina densa the longitudinally oriented autofluorescent fibers of the lamina reticularis (LR) as seen in Figure 2A are visible. Bar: 42 µm. (B) In areas where the lamina densa is missing, the large fibers of the lamina reticularis along with smaller crosslinking fibers are clearly visible (dark arrows). Bar: 10 µm. (C ) Higher magnification shows a network of fine fibrils surrounding the large longitudinally oriented fibers (arrows). Bar: 3 µm.

In preparations where the epithelium was removed with EDTA, basal cells and patches of columnar cells still covered much of the basement membrane similar to that shown in Figure 3A; however, the lamina densa was usually intact. The lamina densa was removed in these preparations with surgical adhesive, revealing a compact lamina reticularis when compared with protease-treated samples. At higher magnifications, it is clear that the fibers are covered with an amorphous substance (Figure 5). This substance was absent in preparations treated with protease, suggesting that it had been digested away by the protease (Figures 4B and 4C).

View larger version (166K): [in this window] [in a new window]   Figure 5.   Scanning electron micrograph of lamina reticularis after EDTA treatment. The fiber framework was found to be covered with an amorphous substance when compared with the fiber framework in Figures 3B and 3C. Bar: 8 µm.

	Discussion

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Previous studies of the basement membrane have identified many components of the lamina reticularis, including the structural proteins present (3, 4, 6). The predominant collagen is type III, followed by collagens I, V, VI, and VII. Nonstructural proteins in the lamina reticularis include tenacin, fibronectin, and proteoglycans. The lamina reticularis is considered to be a specialized extension of the extracellular matrix made up of collagen fibrils, but no particular organization of the fibrils was recognized. However, some TEM studies have demonstrated anchoring fibers (12), "microthread-like filaments" (13), "cords" (14), and organized distribution of sulfated domains (15) within the basement-membrane zone, suggesting that the lamina reticularis has some type of organization. The present studies demonstrate that the collagen fibrils of the lamina reticularis in the rat trachea are arranged as large fibers in a highly organized manner within the basement-membrane zone. The arrangement is in the form of a mat, with the large fibers oriented along the longitudinal axis of the airway. Smaller fibers are crosslinked with the larger fibers to complete this structure. Other small fibers are oriented around the large fibers. Openings in the mat were observed in the lower regions of the trachea; most of these were found near the cartilage rings. The purpose of these openings was not determined; however, it was assumed that the larger openings are associated with glands and the smaller openings with nerves. Openings in the fibrous mesh of the mat also allow for interaction of the underlying attenuated fibroblasts directly with the overlying lamina densa. In the rat trachea there are approximately 7,000 such interactions per square millimeter of epithelium (16).

The structural proteins making up the fibrous framework of the lamina reticularis were not identified in this study. However, according to the literature, the large fibers oriented with the longitudinal axis of the airway probably consist of collagen III with some collagen I and V (4). The fine fibers encircling the large fibers are consistent with descriptions of collagen VI (17, 18). Immunohistochemistry for elastin revealed a banding pattern on the surface of the collagen fibers but not between fibers. This observation indicates that the crosslinking fibers are made up of elastin and probably the elastin-associated microfibrils (fibrillins, microfibril-associated glycoproteins, emilin, fibulin-2) (17). The amorphous proteins covering the fibrous framework probably contain proteoglycans (hyaluronan, chondroitin sulfate, heparin sulfate) and the other nonstructural proteins reported to be in the lamina reticularis (3, 4). Autofluorescence of the lamina reticularis is thought to be due to collagen III and elastin (19).

The significance of the lamina reticularis being arranged as a mat is not clear. This configuration of the fibers could be associated with its role in attachment of the epithelium to the matrix and/or the cylindrical nature of the trachea. Functionally, the lamina reticularis represents the extracellular matrix that lies between the epithelium and a layer of attenuated fibroblasts lining the airways. These tissues are part of an anatomical and functional configuration termed the epithelial-mesenchymal trophic unit (20). The epithelial-mesenchymal trophic unit allows for the localized exchange of information between the epithelium and fibroblasts via the basement-membrane zone during growth and inflammation and in response to injury. The mat-like structure of the lamina reticularis may be a more efficient means of localizing signaling activities and providing directional information between the epithelium and fibroblasts than would a more random structure (1, 21). For example, some signaling activities are carried out by nonstructural proteins (proteoglycans). Proteoglycans bind to collagens and many other molecules and have a number of known functions (22), including regulation of matrix water balance; assembly and maintenance of basement membranes; cell binding, migration, and proliferation; localization of growth factors; and signaling between the matrix and other tissue elements. Conceivably, the mat-like configuration could allow for more spatial and directional organization of proteoglycan-binding sites and thus signaling molecules in the airway. However, at this time, the significance and function of the mat-like structure are just beginning to be studied.

In conclusion, this study has shown that the collagen framework of the lamina reticularis in rat trachea is a complex, highly organized structure. In human large airways the lamina reticularis is the region of the basement-membrane zone that accumulates collagen and leads to the subepithelial fibrosis associated with asthma and other chronic airway diseases (23). How subepithelial fibrosis develops and its effects on the normal functions of the lamina reticularis are not known. Recognizing the complex structure of the lamina reticularis in the rat trachea may help answer these questions in the future.