Porosity of the Basement Membrane Overlying Peyer's Patches in Rats and Monkeys

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1 GASTROENTEROLOGY 1986;91: Porosity of the Basement Membrane Overlying Peyer's Patches in Rats and Monkeys SAMUEL G. McCLUGAGE, FRANK N. LOW, and MARIL YN 1. ZIMNY Department of Anatomy, Louisiana State University Medical Center, New Orleans, Louisiana The porosity of the epithelial basement membrane (basal lamina) overlying lymphoid follicles within Peyer's patches was studied in rats and monkeys by scanning electron microscopy. Basement membranes of lymphoid follicles are markedly porous, more conspicuously so than those of adjacent villus cores. The porosity increases centrifugally from the apex of the follicle to its periphery, where the basement membrane continues into the cul-de-sacs of the crypts. Such porosity may facilitate bidirectional passage of lymphocytes during an immune response. The unique structure of the basement membrane overlying lymphoid follicles suggests a biologic adaptation of this tissue boundary to a specific physiologic activity of the organism. Peyer's patches are known to be a functional part of the gut-associated lymphoid tissue. The epithelium covering the dome of the lymphoid follicles within Peyer's patches plays a specialized role in the uptake and processing of antigen from the lumen of the gut. lt transports antigen from the lumen to underlying lymphocytes and macrophages (1). The protruding domelike surface of the follicle, the presence of M cells of epithelial origin, intraepithelial lymphocytes, and the relative absence of mucus-secreting goblet cells within the follicle epithelium all appear to assist in the trapping and transport of antigen (1-5). These features have been described in humans (4) as well as in other species (1-3). The specialized nature of the follicle epithelium is in marked contrast to the epithelium that covers the surrounding intestinal villi (1-5). Received January 28, Accepted April 14, Address requests for reprints to: Dr. Sam G. McClugage, Department of Anatomy, Louisiana State University Medical Center, 1901 Perdido Street, New Orleans, Louisiana This study was supported by National Institutes of Health grants HL and RR by the American Gastroenterological Association /86/$3.50 The majority of previous studies on the mucosal surface of Peyer's patches emphasize'd the uniqueness of the follicle epithelium (1-5). Little attention has been given to the underlying basement membrane of this epithelium. Although some studies noted the presence of a basement membrane (2,6,7)' the staining quality (contrast) or magnification was not sufficient for accurate morphologic assessment. Recently, a technique has been developed that permits the selective removal of epithelium from its underlying basement membrane (8-10) and the visualization of the latter by scanning electron microscopy. This approach (11) has demonstrated that the underlying basement membrane of intestinal epithelium contains numerous pores of variable size, and that these pores are numerically increased within basement membranes overlying lymphoid follicles. This increased porosity appears to be a morphologic adaptation to the lymphoid follicle, and thus may play an important role in antigen-to-cell and cell-tocell interactions during an immune response in the gut wall. This paper presents a scanning electron microscopic study of the pores within the basement membrane of the follicle epithelium. Materials and Methods Male and female Sprague-Dawley rats, 6 wk to 6 mo of age, were used for this study. These animals were housed in cages and fed standard laboratory food. Two pieces of terminal ileum were obtained from owl monkeys (Aotus trivirgatus) to compare the morphology of their basement membranes with those of rats. The methodology for observing, by scanning electron microscopy, the exposed epithelial basement membrane of the intestine has been reported in detail in an earlier communication (10). Briefly, selected pieces of terminal ileum from Sprague-Dawley rats or owl monkeys were immersed in 1% aqueous boric acid overnight. Boric acid acts as a tissue softener and produces complete dissociation of epithelium from its underlying basement mem-

2 November 1986 PEYER'S PATCH BASEMENT MEMBRANE 1129 Figure 1. Small intestine, rat. Epithelial removal is complete in all figures. Several lymphoid follicles (LF) are located within a Peyer's patch surrounded by villus cores (Ve). The porosity of the basement membrane overlying the follicles is greater than that of the villus cores. Ostia (0) of crypts of Lieberkuhn are also evident. brane (10,12). The tissues were then dehydrated in ascending percentages of acetone (25%, 50%, 75%, 95%, 100%) for at least 10 min each, except for the final step, which requires at least 30 min in pure acetone. Although boric acid usually provides adequate removal of epithelium (i 1), further microdissection of the tissue was usually carried out by placing the tissues in a sonicator (disontegrator, Ultrasonic Industries, Inc., Engineers Hill, Long Island City, N.Y.) at 80,000 cycles/s for 10 min. This treatment completely removed the epithelium but left the basement membrane intact. After sonication, routine preparatory methods were used for scanning electron microscopy. To prepare the specimens for transmission electron microscopy, selected scanning electron microscopic preparations were removed from their studs, immersed in propylene oxide, and embedded in either Epon-Araldite or Spurr's low viscosity embedding medium. Thin sections were stained with uranyl acetate and lead citrate before examination in an AEI-6B electron microscope. Results Removal of the epithelium overlying the lymphoid follicles of a Peyer's patch reveals an underlying basement membrane that exhibits a marked porosity, as demonstrated by scanning electron microscopy (Figure 1). The porosity of the Peyer's patches within the terminal ileal segments of all animals was similar. Such porosity is also evident in solitary lymphoid follicles located in the gut wall (Figure 2). The porosity of the basement membrane overlying the follicles can be easily contrasted with that of the adjacent villus cores, which consist of a basement membrane encircling an interstitial space (Figures 1-3). Occasionally, a villus core is observed sitting directly on the luminal surface of a lymphoid follicle, thereby demonstrating the marked reduction in porosity of the basement membrane as it reflects onto the surface of the villus core (Figure 3). The porosity of each follicle increases centrifugally from the cap to the periphery of the follicle, where the porous basement membrane continues into the cul-de-sacs of the crypts of Lieberkuhn (Figures 1 and 4). The porosity within the basement membrane of the upper edges of the crypts of Lieberkuhn surrounding the follicle is markedly greater than that of the crypts at the base of the villus cores (Figure 4); however, further studies into the deeper parts of the crypts were not performed. Closer examination of the population of pores within the basement membrane from various areas demonstrates much morphologic heterogeneity (Figure 5). Pores have also been demonstrated by transmission electron microscopy (Figure 6). In addition to their obvious differences in size, the ultrastructure of each pore is distinctly different, possibly due to different stages of pore formation or repair, or both. These

3 1130 McCLUGAGE ET AL. GASTROENTEROLOGY Vol. 91, No.5 Figure 2. Small intestine, rat. An isolated lymphoid follicle (LF) between several villus cores (VC). The porosity of the basement membrane is markedly increased on the lymphoid follicles. Ostia (0) of crypts of Lieberkuhn are also evident. Figure 3. Small intestine, owl monkey. A single villus core (VC) projects from an isolated lymphoid follicle (LF). Note that the basement membrane is less porous over the villus core even though it is sitting in the center of a follicle.

4 November 1986 PEYER'S PATCH BASEMENT MEMBRANE 1131 Figure 4. Small intestine, rat. The porosity of basement membrane overlying a lymphoid follicle increases centrifugally from the cap (C) to the periphery, and continues into the ostia (0) of the crypts of Lieberkuhn that surround the follicle. Figure 5. Small intestine, rat. The sizes of the pores within the basement membrane overlying a lymphoid follicle are quite heterogeneous, possibly connoting various stages of pore formation or repair, or both.

5 1132 McCLUGAGE ET AL. GASTROENTEROLOGY Vol. 91, No.5 6.5pm Figure 6. Small intestine, rat. The epithelium covering a lymphoid follicle has been removed, exposing its underlying basement membrane (BM) to the metal coating (MC), a small fragment of which lies within what appears to be the edge of a pore (P). The thickness of the fibrillar substratum is bracketed. The underlying connective tissue fibrils (C) are typical of interstitial tissue in the tunica propria. pores also appear to penetrate the underlying fibrillar substratum (interstitial space). Discussion Basement membranes are found outside the plasmalemma of all histologic cell types except connective tissue cells and their derivatives. Epithelium, central nervous system tissue, muscle, peripheral nerve, and fat each very likely manufactures its own basement membrane composed of collagen, glycoproteins, and proteoglycans. This report demonstrates face-on views of pores within epithelial basement membranes overlying lymphoid follicles in rats and monkeys. Their occurrence is evident in fixed and unfixed, sonicated and unsonicated, tissue samples (11). This porosity increases centrifugally from the apex of the follicle to its periphery, and continues into the upper edges of the cul-de-sacs of the crypts of Lieberkuhn. The porosity of the basement membranes of the surrounding villus cores and their associated crypts of Lieberkuhn is markedly less (11). The biologic significance of the porous epithelial basement membrane overlying lymphoid follicles within the gut wall is of special interest. In the human intestinal epithelium, 20% of the total intraepithelial cell population is composed of nonepithelial cells, mostly lymphocytes (13), and this percentage is even greater within the mucosa overlying lymphoid follicles than anywhere over villi (14). As Peyer's patches are known to be sites of antigen sampling with the overlying epithelium heavily infiltrated by lymphocytes (14), such increased porosity of the basement membrane in these areas is not surprising. The pores are probably formed by migrating lymphocytes inasmuch as similar pores have been described during passage of lymphocytes through the basement membrane of normal skin (15) and of rat intestinal villi (16). The scanning electron micrographs illustrated in this report compare the differences in porosity between the epithelial basement membrane of an intestinal villus core and that of a lymphoid follicle. Such quantitative morphologic differences suggest concomitant differences in mucosal permeability of various parts of the small intestine. In this regard, piglet jejunal mucosal segments containing Peyer's patches demonstrate greater transport of horseradish peroxidase than adjacent patch-free segments (17). The increased transport of the Peyer's patch segments may be due to decreased numbers of goblet cells, thus reducing the mucous barrier, or to the pinocytotic activity of M cells (7). However, the increased transport may also be due to porosity differences between the basement membranes of the patch and patch-free segments. The morphologic heterogeneity of the pore population demonstrated in this report suggests that pores at anyone time are in different stages of repair or

6 November 1986 PEYER'S PATCH BASEMENT MEMBRANE 1133 formation. The epithelial basement membrane of rat intestine is an integral part of the extracellular matrix and may have the ability to flow back together after penetration by cells, cell processes, or various molecular aggregates such as chylomicrons. To maintain its own integrity, the basement membrane most likely closes over the patencies that occur during passage of various substances. In this regard, migrating lymphocytes passing through cutaneous basement membranes produce a sequence of deformations in the basement membrane that is followed after passage by a gradual disappearance of a pore and the eventual repair of the defect (15). The physical and chemical properties of basement membranes that possibly permit pore formation to occur have been discussed in an earlier communication (11). The regional differences in porosity of the intestinal epithelial basement membrane demonstrated in this report and an earlier one (11) support the current belief that basement membranes are important structural components of the extracellular matrix. This report and others (11,18) demonstrate that basement membranes can display a morphologic adaptation by a specific tissue or organ. This adaptation may play an integral role in the physiologic response of an organism. For example, the porous basement membrane overlying lymphoid follicles described in this report probably plays a part in the immune response of the organism. One might speculate that such porosity could facilitate passage of luminal antigens into the follicle and movement of chemical mediators from the follicle into the epithelium. This role heretofore has been largely unrecognized because of the difficulty encountered in adequately studying epithelial basement membranes. References 1. Owen RL, Bhalla DK. Lymphoepithelial organs and lymph nodes. In: Hodges GM, Carr KE, eds. Biomedical research applications of SEM. New York: Academic, 1983: Bhalla DK, Owen RL. Cell renewal and migration in lymphoid follicles of Peyer's patches and cecum-an autoradiographic study in mice. Gastroenterology 1982;82: Owen RL, Nemanic P. Antigen processing structures of the mammalian intestinal tract: an SEM study of Iymphoepithelial organs. In: Becker RP, Johari 0, eds. Scanning electron microscopy. Chicago: SEM Inc" 1978: Owen RL, Jones AL. Epithelial cell specialization within human Peyer's patches: an ultrastructural study of intestinal lymphoid follicles. Gastroenterology 1974;66: Abe K, Ito T. A qualitative and quantitative morphologic study of Peyer's patches of the mouse. Arch Histol Jpn 1977;40: Chin KN, Hudson G. Ultrastructure of Peyer's patches in the normal mouse. Acta Anat (Basel) 1971;78: Owen RL. Sequential uptake of horseradish peroxidase by lymphoid follicle epithelium of Peyer's patches in the normal unobstructed mouse intestine: an ultrastructural study. Gastroenterology 1977;72: Highison GL, Low FN. Microdissection by ultrasonication after prolonged OS04 fixation: a technique for scanning electron microscopy. J Submicrosc CytoI1982;14: McClugage SG, Low FN. Porosity of the intestinal basal lamina demonstrated by microdissection. J Cell BioI 1982: 95:113a. 10. Low FN, McClugage SG. Microdissection by ultrasonication: scanning electron microscopy of the epithelial basal lamina of the alimentary canal in the rat. Am J Anat 1984;169: McClugage SG, Low FN. Microdissection by ultrasonication: porosity of the intestinal epithelial basal lamina. Am J Anat 1984;171: Vial J, Porter KR. Scanning microscopy of dissociated tissue cells. J Cell BioI 1975;67: Toner PG, Ferguson A. Intraepithelial cells in the human intestinal mucosa. J Ultrastruct Res 1971;34: Owen RL, Allen CL, Stevens DP. Phagocytosis of Giardia muris by macrophages in Peyer's patch epithelium in mice. Infect Immunol 1981;33: Warfel KA, Hull MT. Migration of lymphocytes through the cutaneous basal lamina in normal skin: an ultrastructural study. Anat Rec 1984;208: Komuro T. Fenestrations of the basal lamina of intestinal villi of the rat. Scanning and transmission electron microscopy. Cell Tissue Res 1985;239: Keljo DJ, Hamilton JR. Quantitative determination of macromolecular transport rate across intestinal Peyer's patches. Am J PhysioI1983;244:G Carlson EC, Kenney Me. An ultrastructural analysis of isolated basement membrane in the acellular renal cortex: a comparative study of human and laboratory animals. J MorphoI1982;171: