THE ANATOMICAL RECORD 239:297-305 (1994) Ileal Peyer’s Patches in Pigs: Intercellular and Lymphatic Pathways STEWART LOWDEN AND TREVOR HEATH Department of Anatomical Sciences, University of Queensland, St. Lucia, Australia ABSTRACT Background: The lymphatics of Peyer’s patches disseminate immunological information from the gut and thus play a key role in protection of the body against environmental pathogens. The aim of this project was to describe the lymphatic pathways of these Peyer’s patches in pigs, and the mucosal intercellular spaces which lead to these lymphatics. Methods: Ileal tissue from living or freshly killed pigs was examined by light microscopy or electron microscopy, or was injected with Mercox (CL2B, Japan Vilene Hospital, Tokyo) for scanning electron microscopy of corrosion casts. Results: Intercellular fluid between intestinal epithelial cells passes through pores in the basal lamina to mix with that in the intercellular spaces and prelymphatic intercellular channels of the lamina propria and follicle domes. From there, lymph enters lacteals in the villi, or a branching network of vessels within the lamina propria. Small lymphatics penetrate the muscularis mucosae and are continuous with (1) lymphatic vessels which pass directly to the deep submucosa between follicles, or (2) lymphatic sinuses which lie adjacent to the follicles. This differs from the situation in sheep and rabbits. Basal lymphatics beneath the follicles convey lymph to vessels which leave the surface at the serosa. Conclusion:The differences in the structure and arrangement of the lymphatics of Peyer’s patches between pigs, sheep, and rabbits will require further investigation to determine if such variation between species has an effect on the distribution of immune products to effector sites. 0 1994 Wiley-Liss, Inc. Key words: Peyer’s patches, Lymphatics, Intercellular space, Pig, Ileum Peyer’s patches, as part of the gut-associated lym- light or electron microscopy were the main methods phoid tissue (GALT), contribute in a n important way to used. the protection of intestinal mucosal surfaces against MATERIALS AND METHODS infection (Pabst, 1987). The lymphatics of Peyer’s Three male Large White X Landrace pigs, three patches play a key role in these immune reactions and 25-30 kg in weight, were used. In months of age and in disseminating immunological information to other addition, eleven fresh gastrointestinal tracts from pigs body surfaces (McGhee, 1992). Although studies have been made on histological less than six months in age were collected from a local structure, ontogeny, cells, and cell products associated abattoir. The pigs were housed in covered pens and given free with Peyer’s patches in pigs (Sloss, 1954; Chu et al., 1979a,b; Torres-Medina, 1981; Chu and Liu, 1984; access to water. Food was withheld 12 h r prior to surBinns and Licence, 1985; Pabst et al., 1988; Rothkotter gery. They were sedated with intramuscular azaperone et al., 1990), the only detailed information on the lym- (Stresnil, Boehringer Ingelheim, Artarmon, New phatic pathways of Peyer’s patches comes from sheep South Wales), anaesthetised with intravenous thiopen(Lowden and Heath, 1992) and a scanning microscope tone (Pentothal, Boehringer Ingelheim, Artarmon, study on rabbits (Ohtani and Murakami, 1990). Stud- New South Wales), and killed, without regaining conies into the lymphatics of Peyer’s patches in pigs, which sciousness, with a n overdose of pentobarbitone (Lethaplay a key role in their function, seems more important barb, Arnolds of Reading, Boronia, Victoria). considering that pigs are often used a s models to investigate human gastrointestinal disorders. Of further significance, lymph pathways of other lymphoid tissues in pigs, the lymph nodes (Spalding and Heath, 1987, Received June 7, 1993; accepted January 15, 1994. 1989), show marked differences to most other species. Address reprint requests to Stewart Lowden, Department of AnaThe aim of this study was to describe the lymphatics tomical Sciences, University of Queensland, St. Lucia, Australia, of Peyer’s patches in the pig ileum. Mercox casting and 4072. 0 1994 WILEY-LISS, INC. 298 S. LOWDEN AND T. HEATH Fig. 1. Light micrograph of part of an ileal Peyer’s patch stained with hematoxylin and eosin. A follicle dome (D) is surrounded by villi (V), some of which show centrally placed lacteals (LA). Crypts of Leiberkiihn ( G )descend through the lamina propria and end abruptly a t the muscularis mucosae (MM). In the submucosa, a follicular sinus (S) lies adjacent to lymphoid follicles (F).A lymphatic vessel (LV) lies adjacent to the mucosal side of the muscularis externa (ME). I, interfollicular tissue; L, lumen; arrowhead, serosa. x 50. Fig. 2. Light micrograph of a longitudinal mucosal fold in part of a n ileal Peyer’s patch, stained with hematoxylin and eosin. Follicles (F) within the fold are supported by connective tissue (CT) which contains lymphatic vessels (LV), and joins to the muscularis externa (ME). A follicular sinus (S) lies adjacent to a follicle. L, lumen; V, villi; arrowhead, serosa. x 40. In the anaesthetised pigs, lymphatic pathways in the intestinal wall were distended by ligating the intestinal lymphatic trunk until the mesenteric lymphatics were visibly distended, or by partially ligating the cranial mesenteric vein to increase net movement of fluid out of capillaries in the intestinal wall. The mesenteric lymphatics and the blood vessels supplying the ileum were then ligated, and sections of the ileum were tied off. The tissue for light microscopy and scanning electron microscopy was fixed by injecting 4% paraformaldehyde into the lumen then removing the ileum and placing it in the same fixative. Six to ten equidistant sections were cut from each specimen, then dehydrated in alcohol, cleared in xylol, and mounted in wax. Histological sections were prepared a t 8 pm thickness and stained with hematoxylin and eosin. Specimens were photographed using a n Olympus PM-1OAD photographic system attached to a n Olympus BHT binocular microscope. Tissue for scanning electron microscopy was dehydrated with ethanol, cleared with xylol, and mounted in wax. A face was prepared with a microtome. The sections were de-waxed, then dried by the critical point method and examined using a JSM 6400F scanning electron microscope. Tissue for transmission electron microscopy was fixed with a solution of 4% paraformaldehyde and 2.5% glutaraldehyde. This was injected into the lumen, then the ileum was removed and stored for 24-48 h r in the fixative. Sections were postfixed in 1%osmium tetroxide, stained in 5% uranyl acetate, then dehydrated and embedded in AralditeIEpon mixture. Thick sections LYMPHATICS OF PIGS' PEYERS PATCHES TABLE 1. Distribution of mucosal folds and the lymphoid follicles - Folds per section ( n l = 33) Folds containing follicles per section ( n l = 33) Follicles per fold (n2= 74) Average 5.3 Range 0-10 2.2 7.5 0-6 1-15 'Where n = total number of cross-sections examined. 2Where n = total number of folds containing follicles. Fig. 3. Scanning electron micrograph of a Mercox cast which has filled the intercellular spaces (IE) between the epithelial cells of villus. Towards their apices, these casts narrow and form a series of interconnecting pillars (P) which contain small holes (arrows). The luminal surface (asterisk) is roughened but uniform. These casts are joined to intercellular casts of the lamina propria (IL) by short connections (C). BL, basal lamina space; L, lumen. X 1,400. Fig. 4. Scanning electron micrograph of the basal lamina (BL)of the villus after removal of the epithelial cells with 1%boric acid. Rounded holes are present on the surface. x 1,300. 299 were stained with toluidine blue, and ultrathin sections were mounted on uncoated copper grids, stained with uranyl acetate followed by lead citrate, and examined with a Zeiss 10A-B electron microscope. Casts of the lymphatic pathways were made in both living and abattoir specimens. One ml of undiluted Mercox (CL-BB, Japan Vilene Hospital, Tokyo) was injected into the interstitium of the follicular region of the ileal wall using a 26G needle attached to a hand- Fig. 5. Scanning electron micrograph of the basal lamina of a follicle dome (D) joining that of adjacent villi (V) between the crypts of Leiberkuhn (G).The holes in the basal lamina of the dome are larger and more abundant towards the edge of the dome (arrow). X 180. Fig. 6. Scanning electron micrograph showing holes (HI in the basal lamina (BL) of a follicle dome. A cell (arrow) can be seen passing through one of these holes. Some dome epithelium (DE) is still attached to the basal lamina. x 1,300. 300 S. LOWDEN AND T. HEATH held syringe until casting material appeared in the serosal lymphatics (usually 30-45 sec). The ileum was then washed in warm water (60°C) for 2 hr, and digested in 4 changes of 15% NaOH. Casts were washed and sonicated in distilled water, dried and sputter coated with platinum, and viewed with a JSM 6400F SEM a t a n accelerating voltage of 2.0-2.5 kV. The intestinal epithelial cells were removed to expose the basal lamina using boric acid in a technique modified from Low and McClugage (1984). Ileal tissue from abattoir specimens was immersed in 1% aqueous boric acid for 18 hr, fixed in 10% neutral buffered formalin for 8-12 hr, washed and sonicated in distilled water, then dehydrated in a graded series of alcohols and dried by the critical point method. Specimens were sputter coated with gold and viewed with a JSM 6400F SEM. RESULTS Light microscopy was used to examine Peyer’s patches from 33 cross sections of ileum taken from seven pigs (Fig. 1).These patches formed a continuous band along the antimesenteric border of the ileal wall. Longitudinal folds, which were visible grossly on the mucosal surface of the ileum, often occurred along this antimesenteric border and contained follicles (Fig. 2). The distribution of these mucosal folds and the lymphoid follicles within these folds is shown in Table 1. Fig. 7. Scanning electron micrograph of a Mercox cast showing the intercellular and lymphatic pathways associated with a follicle dome. The intercellular casts of the dome (asterisk) and lamina propria (star)are continuous with a network of intercellular channels (arrowheads) which are less than 11 Fm across. These enter lymphatic vessels (LV) which form a complex branching network in the lamina propria around the follicle domes. x 100. Intercellular Spaces When Mercox was injected into the follicular region of the Peyer’s patches, it filled intercellular spaces and lymphatic vessels and sinuses. For example, Mercox filled the spaces between epithelial cells on the shaft of the villus, forming casts that were 16-26 pm in height and had a smooth surface (Fig. 3). Towards their apices, these casts narrowed, often forming a series of interconnected pillars. The luminal surface was roughened but formed a n even plane. Discrete round or oval holes ranging from 160 to 500 nm in diameter were prominent, especially in the apical portion. Laterally, Mercox appeared to move freely between cells forming a continuous interconnecting network (Fig. 3). These epithelial casts were separate from the underlying intercellular casts of the lamina propria. They were, however, joined by short, straight connections 5.0-9.0 pm in length and 1.6-6.3 pm in diameter (Fig. 3). Rounded holes of 0.6-2.9 pm diameter occurred in the basal lamina underlying the villus epithelium of samples treated with boric acid (Fig. 4). The basal lamina underlying the dome epithelium showed a greater porosity than that underlying the villus epithelium, with holes ranging from 2.0-9.0 pm in diameter. These holes were larger and more abundant towards the edge of the dome where the basal lamina was continuous with that of the villus base around the ostia of the crypts of Leiberkuhn (Fig. 5). Stromal tissue was visible beneath these holes, and the occasional lymphoid cell was seen passing through (Fig. 6). Intercellular Channels Casts of the intercellular spaces of the dome and surrounding lamina propria co-existed with, and were often continuous with, a fine branching network of tubu- lar casts ranging in diameter from 3.3-11 km (Fig. 7). Similar casts found beneath the villus epithelium were continuous with the epithelial casts. Impressions of valves were not evident in these tubular casts, and no endothelial cell walls could be identified by transmission electron microscopy. Lymphatic Vessels of the Mucosa Lacteals were single, blind-ending vessels of 11-40 pm in diameter. They were centrally located within villi and were continuous with the lymphatic vessels at the base of the villi. Valves were seen in these mucosal lymphatics (Fig. 8). The endothelium was smooth, but occasionally in the apical or mid-portion of a lacteal, a round hole was seen which appeared to open into the surrounding stromal tissue (Fig. 9). In a cross sectional Mercox cast of a villus, a n intercellular cast was connected to a large mass of Mercox, centrally placed within a lacteal (Fig. 10). The lacteals and the tubular casts then joined larger lymphatic vessels in the lamina propria. These vessels, 14-50 pm in diameter and containing valves, formed a complex branching network around the follicle domes and crypts of Leiberkuhn (Fig. 7). Lymphatic Pathways of the Submucosa The vessels of the lamina propria penetrated the muscularis mucosae and entered the interfollicular tissue of the submucosa. They followed one of two courses on their way to vessels at the base of the follicles (Fig. 11). Vessels following the first course passed directly to the base of the follicles within the interfollicular connective tissue (Figs. 11, 12). These vessels ranged in 301 LYMPHATICS OF PIGS PEYERS PATCHES Fig. 8.Scanning electron micrograph showing a valve (V) in a lymphatic vessel of the lamina propria. BV, blood vessel; L, lumen. x 1,300. diameter from 16-48 pm. Transmission electron microscopy showed that these vessels had a continuous endothelial lining which was supported by collagen bundles in various orientations, and fibroblasts with prominent cytoplasmic processes (Fig. 13). This tissue merged with the surrounding connective tissue. Smooth muscle cells were not common. The vessels which followed the second course lay adjacent to the follicles, forming a n anastomosing network around their apical portion. These vessels had a diameter of 10-30 pm (Fig. 12). Some of these vessels then joined the first group, whilst others, in coursing to the base of the follicles, lay against the follicle capsule forming follicular sinuses. These sinuses were up to 140 pm in width, but did not completely surround the follicles (Figs. 11, 12). The sinus wall adjacent to the interfollicular tissue was lined by a more convoluted endothelium than seen in the first group of vessels, but generally showed a similar arrangement of collagen and fibroblasts (Fig. 14). Towards the base of the follicles, nerve ganglia and nerve bundles were often seen in close association with this endothelium. The sinus wall adjacent to the follicle capsule was lined by a flattened endothelium and supported by tightly packed layers of collagen and fibroblasts. This connective tissue formed the bulk of the follicle capsule. It separated the sinus from the lymphoid tissue of the follicle (Fig. 15), or in the absence of a sinus, merged with the surrounding interfollicular tissue. The follicular sinuses then joined with the first group of vessels, narrowing to less than 50 p,m diameter, before reaching the base of the follicles. No lymphatic vessels were seen entering or leaving the follicles themselves. These lymphatics draining the interfollicular region often converged and joined before entering larger basal vessels, 40-170 p m diameter, which ran perpendicular to them. These basal lymphatics, which contained prominent valves, ran adjacent to, or up to 170 pm beneath the follicles (Figs. 11, 16). The endothelium was supported by collagen and fibroblasts and was surrounded by smooth muscle cells. Smooth muscle cells appeared less intimately associated with the endothe- Fig. 9. Scanning electron micrograph of a lacteal (LA) within a villus (V). A hole (arrowhead), 12 pm across, is present in the smooth endothelial lining (E) of the lacteal (LA). L, lumen. x 500. Fig. 10.Scanning electron micrograph of a Mercox cast showing a cross-section of a villus. Casts of the intercellular spaces between epithelial cells (IE) are continuous with intercellular casts (IL) and intercellular channels (arrowheads) of the lamina propria by short connections (C). In turn, these casts are continuous with a large mass of Mercox, centrally placed within a lacteal (LA). x 450. lium of the lymphatics in comparison to the smooth muscle cells of the blood vessel wall (Fig. 17). These basal lymphatics coursed beneath the follicles, carrying lymph away from the local follicular environment before entering vessels draining the serosal surface (Fig. 11). DISCUSSION In the pig, intercellular fluid in the ileal epithelium passes through pores in the basal lamina to mix with 302 S. LOWDEN AND T. HEATH Fig. 11. Scanning electron micrograph of a Mercox cast showing the submucosa of an ileal Peyer’s patch. Lymph vessels (arrow) lie adjacent to the cup-like follicle spaces (asterisks), or course between them (arrowheads). All vessels join the basal lymphatics (B) running perpendicular to them beneath the follicles. I, interfollicular connective tissue casts. x 40. Fig. 12. Scanning electron micrograph of a Mercox cast showing the submucosa from the space occupied by the muscularis mucosae (MM) to a basal lymphatic iB) beneath the follicle space (double-headed arrow). Lymph vessels (LV) pass directly to the basal lymphatic within the interfollicular tissue. Other vessels (arrows)form a n anastomosing network around the apical portion of the follicle. Some branches, in coursing to the base of the follicles, lie against the follicle x 60. capsule forming follicular sinuses (3. that in the intercellular spaces and prelymphatic intercellular channels of the lamina propria and follicle domes. From there, it enters the lymphatics: lacteals in the villi and a branching network of lymphatic vessels in the lamina propria, vessels passing between the follicles and sinuses adjacent to the follicle capsule, and basal lymphatics coursing parallel to the serosal surface beneath the follicles. Some histological features of ileal Peyer’s patches in pigs and their anatomical location have been described previously (Sloss, 1954; Chu et al., 1979a,b). However, the permanent mucosal folds which contain variable numbers of follicles, are not the “plicae circulares” described by Sloss (1954), for when they are present, they form longitudinal folds. These folds more closely resemble the laminar folds of the large intestine of the koala (Hanger and Heath, 1994) and may provide additional surface area for the uptake of nutrients and antigens. Mercox entered the spaces between the epithelial cells from the lamina propria by passing retrogradely through the pores in the basal lamina. Although filling these epithelial spaces with Mercox would have distended them, these casts provided a three-dimensional representation of the extent of the spaces and possible routes of fluid movement between the cells. Apically, the casts narrow, form pillars, and end abruptly. These restrictions on Mercox flow appear to be the result of occluding junctions and belt desmosomes which join the most apical portion of the lateral cell membranes (Madara and Trier, 1987; Gebert and Bartels, 1991). These attachment zones appear to occur at a uniform level across the epithelium a s represented by the even plane of the casts on the luminal surface. The discrete holes which appear sporadically, usually in the apical portion of these epithelial casts, may represent sites of intercellular attachment by spot desmosomes. This is supported by the fact that these holes are of a similar diameter to the spot desmosomes described by Madara and Trier (1987). Although some distortion and shrinkage of Mercox occurs during polymerisation (and this cannot be overlooked when examining such holes at high magnification), the evidence suggests that these changes are minimal (Lametschwandtner e t al., 1990). The intercellular spaces between epithelial cells are continuous with those of the lamina propria through pores in the basal lamina. These pores underlying the villus epithelium in the small intestine of pigs are similar to that described in rats (Low and McClugage, 1984; McClugage and Low, 1984; Komuro, 1985; McClugage et al., 1986; Sugimoto and Ogata, 1989). The diameter of the pore casts are larger than that of the pores seen in samples treated with boric acid, possibly due in part to the pressure of Mercox distending them. Studies in rats have indicated that these pores probably represent a transitional phase of deformation in the basal lamina (McClugage and Low, 1984; Komuro, 1985) permitting the passage of cells, cell processes, and molecular aggregates (McClugage and Low, 1984). This study suggests that these pores may also provide a significant route for the passage of intercellular fluid between intestinal epithelium and the lamina propria. In pigs, as in rats (Low and McClugage, 1984; McClugage and Low, 1984; McClugage ei d . , 1986), the basal lamina underlying the villus epithelium was continuous with that underlying the dome epithelium of the ileal Peyer’s patch. This basal lamina of the follicle dome was also similar to rats in having a greater porosity than the basal lamina of the villus, and a heterogeneous collection of pores of similar dimensions LYMPHATICS OF PIGS PEYERS PATCHES 303 which tend to increase centrifugally from the apex of the dome to the edge between the ostia of the crypts (McClugage et al., 1986). In pig, as in human (Kihara et al., 1991) and rabbit Peyer’s patches (Ohtani e t al., 1991), lymphoid cells can often be seen projecting through the pores in the basal lamina from the follicle domes. These pores appear to be important sites of lymphocytic movement between the epithelium and the lamina propria. The small tubular, or slightly flattened, branching casts found in the lamina propria and the follicle domes, could be confused with blood capillaries, which are of a similar size (4-10 pm in diameter) (Castenholz, 1989). However, these casts join to the intercellular spaces, and Mercox cannot enter intact blood vessels from the intercellular space (Berens v. Rautenfeld and Wenzel-Hora, 1985).No evidence could be found of endothelial lining or valves, and they were classified as prelymphatic intercellular channels (Castenholz, 1989). Lacteals in the villi are similar to those described in sheep (Lowden and Heath, 1992) in being single, centrally placed, blind ending vessels with a smooth endothelium. However, they contain openings to the surrounding stroma. These may be the pore-like openings described by Berens v. Rautenfeld et al. (1988) which occur in most lymphatic vessels and appear in response to a n increased filling pressure, as may have occurred in these lacteals. Consequently, it is difficult to estimate accurately the dimensions of these lacteals under normal physiological conditions. Lymph from the lacteals and prelymphatic tissue channels enters a series of interconnecting vessels in the lamina propria. These resemble the lymphatic sinuses of the lamina propria of sheep (Lowden and Heath, 1992) in surrounding the follicle domes and the crypts, but they contain valves which are not seen in sheep (Lowden and Heath, 1992). These vessels may carry antigenic materials, cells, and cell products from the follicle dome, or the intercellular fluid, throughout the lamina propria. In most respects, the organisation of the lymphatic pathways in the follicular region of pig Peyer’s patches more closely resembles that of rabbits (Ohtani and Murakami, 1990) than of sheep (Lowden and Heath, 1992). As in rabbits, vessels in the lamina propria are continuous with vessels in the interfollicular tissue through the muscularis mucosae; follicular sinuses only partially surround the follicles, and lymph is conveyed to a Fig. 13. Transmission electron micrograph of the wall of a lymphatic vessel in the interfollicular tissue. The vessel is lined by continuous endothelium (E),and supported by collagen bundles in various onentations ( C ) and fibroblasts with prominent cytoplasmic processes (F). L, lumen. x 2,300. Fig. 14. Transmission electron micrograph of the portion of the wall of a follicular sinus which is supported by interfollicular tissue. The sinus is lined by a continuous convoluted endothelium (E) with prominent, nuclei, and supported by collagen bundles (C) and fibroblasts (F).A nerve bundle (N) lies close to the sinus lumen (L). x 2,300. Figs. 13-15. Fig. 15. Transmission electron micrograph of the portion of the wall of a follicular sinus which is supported by follicle capsule. The sinus is lined by continuous endothelium (E) with tightly packed layers of collagen (C) and fibroblasts (F) lying between the sinus lumen (L)and the lymphoid tissue (LY). x 2,300. 304 S. LOWDEN AND T. HEATH Fig. 16. Light micrograph showing the follicular region of the an ileal Peyer’s patch stained with toluidine blue. A lymphatic vessel (LV), containing prominent valves (V), courses beneath a follicle (F). x 30. series of collecting vessels containing valves a t the base of the follicles. Unlike rabbits, however, where all lymph must flow within sinuses around the follicles (Ohtani and Murakami, 1990), in pigs, some lymph may bypass these sinuses by entering the vessels which course between follicles to the collecting vessels at their base. As lymphoid cells derived from the follicles are known to leave Peyer’s patches via these basal lymphatic vessels (McGhee, 1992), the significance of this difference for each species on the distribution of immune cells to effector sites is unknown. Sheep lymph may also bypass the follicular sinuses by entering septa1 vessels (Lowden and Heath, 1992), but few other similarities exist. For example, valves feature prominently in the lymph pathways associated with Peyer’s patches in pigs, but not in sheep or in rabbits. At the ultrastructural level, the follicular region of pig Peyer’s patches resembles that of sheep (Lowden and Heath, 1992), with lymphatic sinuses and vessels being lined by a convoluted endothelium which is supported by connective tissue elements (but few muscle cells), and the follicle capsule consisting of tightly packed layers of collagen and fibroblasts. Unlike sheep, however, the connective tissue occupies most of the area between follicles, attaching to the follicles at all sites save where a sinus lies laterally against the capsule. This suggests that pigs have a more rigid structure in the follicular region when compared with sheep. The general absence of smooth muscle cells around lymphatic vessels in the follicular region suggests that contraction of the vessel wall is not essential for propelling lymph into the basal collecting vessels. With valves to prevent retrograde flow, contractions of the muscularis mucosae and the muscularis externa may combine to propel lymph into the basal collecting vessels, and through the muscle fibers of the muscularis externa to subserosal vessels. Fig. 17. Transmission electron micrograph of a basal lymphatic vessel (LV) lying adjacent to a blood vessel (BV) in the submucosa beneath the follicles. The lymphatic endothelium (E) is supported by collagen bundles (C) and fibroblasts (F). 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