Distribution of lymphoid tissue in the caecal mucosa of chickens

J. Anat. (1998) 192, pp. 293 298, with 5 figures Printed in the United Kingdom 293 Short Report Distribution of lymphoid tissue in the caecal mucosa of chickens HIROSHI KITAGAWA, YOSHITOSHI HIRATSUKA, TOMOHIRO IMAGAWA AND MASATO UEHARA Department of Veterinary Anatomy, Faculty of Agriculture, Tottori University, Japan (Accepted 8 January 1997) ABSTRACT In order to clarify the fundamental structure of the host defence mechanism in chicken caeca, a detailed analysis of the distribution of lymphoid nodules (LNs) was carried out on longitudinal sections of both the mesenteric (side of the ileocaecal ligament) and the antimesenteric mucosa. An overwhelming majority of solitary or aggregated LNs were located in the mesenteric mucosa, although a few were also found in the antimesenteric mucosa. Of the total LNs, 45 7% were detected at the proximal 7 8% section in the caecal tonsil. LNs (21 4%) were also concentrated in the distal 22 0% section corresponding to the apex. A moderate concentration of LNs (13 1%) was found at the transitional 20 0% region between the base and body. Approximately 80 2% of total LNs were found at the above 3 regions in the mesenteric mucosa. In many cases, the frequency of LNs in the caecal tonsils was opposite to that at the apices. Aggregated LNs were mainly found in the caecal tonsils, transitional region and apex. Almost all aggregated LNs consisted of fundamental nodular units possessing M cells in their follicle associated epithelia. The aggregated LNs in the above 3 regions therefore could provide immunological surveillance against caecal luminal contents. In particular, the cooperative function between LNs of the caecal tonsil and apex might be highly important in maintaining the caecal microenvironment. Key words: Gastrointestinal tract; gut-associated lymphoid tissue; M cells. INTRODUCTION Both the bodies and the apices of galliform caeca allow the residence of uric acid degrading bacteria (Mead, 1989). A number of microorganisms, totalling 10 g (wet weight) exist in the lumen (Mead, 1989). To regulate the proliferation of the microflora continuously and to prevent invasion by extracaecal microorganisms, it is hypothesised that the caecal environment should be maintained by a welldeveloped immunodefence mechanism, such as the organisation of enormous lymphoid nodules (LNs) throughout the mucous membrane. In the past, most studies on chicken gut-associated lymphoid tissue (GALT) have focused on caecal tonsils (Glick et al. 1981; Kato et al. 1992). To date, however, there has been no research on LN distribution in the caeca of chickens. The follicle associated epithelia (FAE) of LNs have the special duty of actively taking up luminal particulates. Therefore, the FAE has also been investigated as an advantageous site for administration of antigens (Eldridge et al. 1990) and some drugs (Michel et al. 1991) with nonbiodegradable properties. Due to the presence of antiperistalsis of the large intestine (Yasukawa, 1959), cloacally administered substances are presumed not to be affected by digestive processes and to be absorbed by caecal FAE. Therefore, LNs in the caecum are considered an important site both for immune responses and medicinal therapies. Thus further knowledge of the distribution of lymphoid tissues might contribute to Correspondence to Associate Professor Hiroshi Kitagawa, Department of Veterinary Anatomy, Faculty of Agriculture, Tottori University, Tottori 680, Japan.

294 H. Kitagawa and others the ongoing research on caecal immune responses. Accordingly, we have made a detailed analysis of the distribution of LNs in chicken caeca. MATERIALS AND METHODS Animals A total of 19 female White Leghorn chickens aged 6 mo were used. Spot checks using a radial immunodiffusion method demonstrated the normoglobulinaemic nature of these animals. Pathological examinations were used to check for signs of infection by parasites or any other pathogenic microorganisms. The specimens were extracted after cervical exsanguination under anaesthesia with pentobarbital sodium. Gross morphometry Twenty eight caeca were extracted from 14 animals and gently straightened on a flat plane. The full length of each caecum was measured using slide calipers, as were the lengths of the caecal tonsil, base, body, and apex. Differences between the right and left caeca were examined statistically. The relative ratios of each caecal segment were used in the estimation of LN distributions. Light microscopy The caeca of 5 animals were cut at the ileocaecal junctions and separated from the mesenteries and iliocaecal ligaments. Each cecum was then carefully rolled on a metal plate with small densely arranged holes. The ligament attaching surface was situated at the central side of the osculating surface and the maximum diameter of rolled intestine was laid at the same level. The rolled caecum on the metal plate was gently covered with another plate and immersed in neutralised 10 or 20% formalin for 7 or 3 d, respectively. Following embedding in paraffin, tissue blocks were sectioned in the plane of the maximum diameter of the intestine. The sections 5 7 µm were stained with haematoxylin-eosin. The locations of secondary LN accompanied by germinal centres were plotted on a low magnification micrograph of a section of a whole caecum. The full length of the caecum was measured along the tunica serosa on the micrograph. The frequency of LNs was estimated in percentage portions of the full caecal length. Paired Student s t tests were employed throughout; statistical significance was accepted at the 5% level. RESULTS Gross anatomy The average lengths of right and left caeca were 14 6 cm (S.D. 1 4 cm, 16 2 cm, X min 12 0 cm) and 14 2 cm(s.d. 1 4 cm, 17 8 cm, X min 12 8 cm), respectively. No significant difference was found between the average lengths of right and left caeca. The average length of all caeca was 14 4 cm. The proximal portion of the caecum from the ileocaecal junction was a narrower tube corresponding with the base. The average lengths of these portions of the right and left caeca were 5 6 cm (S.D. 0 7 cm, 6 7 cm, X min 4 1 cm) and 5 7 cm(s.d. 0 7cm, 7 2 cm, X min 4 4 cm), respectively. The average length of all bases was 5 7 cm or 39 6% of the section of total caecal length. The initial portion of each base included a slightly swollen area suggestive of a caecal tonsil. The average lengths of the right and left caeca were 1 1 cm (S.D. 0 2 cm, 1 6 cm, X min 0 9 cm) and 1 1 cm (S.D. 0 2 cm, 1 4 cm, X min 0 7 cm), respectively. The average length of all caecal tonsils was 1 1 cm or 7 8% of the total caecal length. The distal portion of the caecum was a thicker tube which was further subdivided into a body and apex by a faint constriction. The average lengths of the right and left apices were 3 3 cm(s.d. 0 5 cm, 3 7 cm, X min 2 6 cm) and 3 0 cm (S.D. 0 5 cm, 3 5 cm X min 2 0 cm), respectively. The average length of all apices was 3 2 cm, or 22 0% of the total caecal length. The remaining 38 4% section constituted the body of the caecum. No significant differences were found between the average lengths of each section of the right and left caeca. Light microscopy Almost all aggregated LNs had the same fundamental structure, consisting of nodular units (Fig. 1). Each nodular unit was demarcated by narrow loose connective tissues. A fossula was observed in each nodular unit; the fossula was located centrally at a varying depth and was surrounded by subepithelial lymphoid tissue with peripherally situated germinal centres. Several intestinal crypts originated from each fossula towards the peripheral margin of the nodular units (Fig. 2). Epithelial mitoses were not observed in the fossulae but were frequent in the intestinal crypts. Almost all LNs were covered by FAE. M cells existed in the lateral FAE or in the epithelium of fossulae. The M cells generally possessed a slightly

Chicken caecal lymphoid tissues 295 Fig. 1. Moderately developed aggregated lymphoid nodules of a caecal tonsil. Note the composition of 3 nodular units (ND). Several intestinal villi are visible on the nodular units. Bar, 200 µm. Fig. 2. A nodular unit of a caecal tonsil. The unit is demarcated by thin connective tissue. The fossula (F) is centrally situated in the unit. Intestinal crypts (arrowheads) originate from F. Germinal centres (asterisks) are positioned at the periphery of the unit. Bar, 100 µm. Fig. 3. (a) M cells and (b) microvillous epithelial cells in the fossula of a caecal tonsil. Note the higher nuclear position (arrows) of M cells and the harbouring of more intraepithelial lymphocytes than in (b). Striated borders are scarcely visible in M cells, whereas microvillous epithelial cells possess distinct striated borders (arrowheads). Bar, 10 µm. Fig. 4. An aggregated lymphoid nodule at its apex. The nodule shows a typical nodular unit. The shallow and wide fossula (F) is formed by bilateral protruded mucous folds. Asterisks, germinal centres, Bar, 100 µm. lighter cytoplasm and a larger nucleus, which was situated above the nuclear level of ordinary columnar epithelial cells. At the apical portion of M cells, the striated borders characterised by intensely acidophilic thin lines were scarcely visible. The basal portion of these cells contained many migrating cells (Fig. 3). The caecal tonsil showed an irregular and protuberant appearance. In moderately developed caecal tonsils, typical nodular units were apparent, although the demarcation of each nodular unit could hardly be recognised in well developed tonsils. Intestinal villi remained on the nodular units in moderately developed tonsils, but not in well developed tonsils. Aggregated LNs were also frequently detected at the transitional region between base and body, and at the apex. In many cases, the fossulae were shallow and wide in nodular units at the apex (Fig. 4) and the germinal centres occasionally protruded into the tunica muscularis at the apex. The total number of LNs both in mesenteric (ileocaecal ligament side) and antimesenteric mucosa in longitudinal sections of right and left caeca was 183 2 (S.D. 45 8, 235, X min 105) and 169 8 (S.D. 78 8, 318, X min 81), respectively. There was no significant difference between the means of the left and right caeca. The total number of LNs in both mesenteric and antimesenteric mucosa was 176 5 (S.D. 64 8, 318, X min 81) in a longitudinal section of the caecum. The majority of solitary and aggregated LNs were distributed in the mesenteric side of the mucous membrane, although a few LNs were also dispersed throughout the antimesenteric

296 H. Kitagawa and others Fig. 5. Distribution of lymphoid nodules in mesenteric (a) and antimesenteric mucosa (b) of chicken caeca. Significantly larger numbers of lymphoid nodules are distributed in mesenteric than in antimesenteric mucosa. In the mesenteric mucosa, concentrations of lymphoid nodules are visible at the proximal and distal portions, and the portion at around 30%. Each bar represents the total number of lymphoid nodules of 10 caeca per 1% section. The length from orifice to apex of a caecum represents 100%. side (Fig. 5). The total number of LNs in mesenteric and antimesenteric mucosa was 129 4 (S.D. 45 1, 222, X min 71) and 46 5 (S.D. 26 7, 96, X min 10), respectively, on longitudinal section. These means were significantly different P 0 001). The total numbers of LNs in the mesenteric mucosa of the right and left caeca were 139 0 (S.D. 35 9, 177, X min 71) and 122 2 (S.D. 52 4, 222, X min 71), respectively. There was no significant difference between the average values of the right and left caeca. In mesenteric mucosa, the proximal 7 8% section corresponding to the caecal tonsil contained 59 1 LNs (S.D. 29 1, 10.5, X min 18), or 45 7% of the total LNs. The distal 22 0% section corresponding to the apex also contained 27 7 LNs (S.D. 19 2, 78, X min 5), or 21 4% of the total LNs. 17 LNs (S.D. 12 0, 38, X min 6), or 13 1% of the total, were located in the proximal 25 0 45 0% section corresponding to the transitional region between base and body. LNs were significantly more numerous (80 2%) in the above 3 regions than in the other regions (19 8%) (P 0 05). No significant differences were found between the means of the above sections of the right and left caeca. Fig. 6. A standard distribution pattern and 2 nonstandard distribution patterns of lymphoid nodules (LN) in the caecal mesenteric mucosa. (a) A frequently recognised distribution pattern. The pattern is similar to that in Fig. 5a. (b) This pattern is characterised by prominent development of LN in the proximal portion but poor development distally. (c) This pattern is opposite that in (b). The LN are highly developed in the distal portion but poorly proximally. Each bar represents the number of LN in a 5% section of the caecum. The length from orifice to apex of the caecum represents 100%. Nonstandard patterns of distribution were found in 6 caeca. In 3 caeca, the ratios of LN number to the total at the caecal tonsil and the apex were 80 8% (S.D. 8 9%, 93 4%, X min 73 9%) and 19 2% (S.D. 8 9%, 26 1%, X min 6 6%), respectively. The ratio in the caecal tonsil was significantly higher than that in the apex (P 0 01). In the other 3 caeca, the ratios of LN number to the total at the caecal tonsil and the apex were 37 0% (S.D. 7 9%, 45 4%, X min 26 4%) and

Chicken caecal lymphoid tissues 297 63 0% (S.D. 7 9%, 73 6%, X min 54 6%), respectively. The ratio in the caecal tonsil was significantly lower than that in the apex (P 0 05). The ratio in the caecal tonsil of the former caecum was significantly higher than that of the latter one (P 0 01), and the ratio in the apex of the former was significantly lower than that in the latter (P 0 01) (Fig. 6). DISCUSSION Glick et al. (1981) reported that the caecal tonsil is a cluster of fundamental uses. In this study, the epithelial cells of the fossula corresponding to the central crypt of Glick et al. showed no mitotic activity and possessed typical M cells. The proliferation site of epithelia seems to be restricted to the intestinal crypt both in the mammalian (Klein & McKenzie, 1983) and avian intestine (Imondi & Bird, 1966; Lilja, 1987). It has never been reported that the epithelia of the intestinal crypt possess M cells either in mammalian or avian species. Therefore, the fossula in aggregated LNs must correspond to a portion of the FAE, but not to an intestinal crypt. Glick et al. (1981) considered that the structural design of nodular units corresponds to that of the mammalian palatine and lingual tonsils. The lymph node tissues of pigs are also made up of nodular units in a similar structural design (Hoshi et al. 1986). In the above mentioned organs, external antigens flow into the central spaces in the nodular units. Therefore, the fact that, in the present study, almost all aggregated LNs of chicken caeca were composed of nodular units suggests that aggregated LNs of chicken caeca are also suitable for the active capture of luminal antigens for immunological surveillance. Caecal tonsils are the major lymphoid tissue in the caecum (Muthmann, 1913). In this study, 45 7% of caecal LNs were distributed in the proximal 7 8% of the caecum. Because the chicken caecum takes in the backflowing urine through the rectum from the urodeum of the cloaca (Braun & Campbell, 1989), it is evident that the proximal portion of the caecum is confronted with continued invasions of bacterial or nonbacterial antigens of extracaecal origin. Therefore, these lymphoid tissues could play a highly important role in immunological surveillance against foreign microorganisms. On the other hand, 13 1% of caecal LNs were detected at the transitional region between base and body in this study. In duck caeca, LNs are also distributed densely in the proximal 10% of the caecal body (Kitamura et al. 1976). The transitional region between the base and body of the caecum in chickens coincides with the proximal 10% of the caecal body in ducks. Because the transitional region between base and body was situated at the proximal end of a fermentation chamber, the surveillance and maintenance of the internal environment of the proximal portion of the chamber might be attributed to LNs of the transitional region. At the extremity of the apex, apical caecal diverticula (ACD) are recognised visually as dome-like protrusions occurring with high frequency in White Leghorn chickens (Kitagawa et al. 1996). The ACD wall displays well developed aggregated LNs. ACD are considered to arise because of the development of LNs and also because of the fragility of the tunica muscularis and the frequent antiperistalsis at the apex (Kitagawa et al. 1996). The present findings of highly developed lymphoid tissues in the apex, as well as the protrusion of their germinal centres into the tunica muscularis, confirm one of the causes for the formation of ACD, and also suggest that the apex is an important site for immunological surveillance in comparison with the caecal tonsil. In Eimeria tenella-infected chickens, LNs were consistently found in the base of caeca approximately 3 cm from the ileocaecal junction (Del Cacho et al. 1993). Among the normal chickens of the present study, no clustering of LNs could be regularly recognised at the corresponding site. From this we presume that LN clusters (Del Cacho et al. 1993) are formed exceptionally in response to particular parasitic antigens, and that exceptional appearances of LNs are due to newly arising antigenic stimulation. Therefore, the present negative correlation between the number of LNs at the caecal tonsils and that at the apex may suggest that their developmental degrees are reflected by timely immune responses in the caecum, and that the caecal tonsil and apex are equally important sites in the caecal host defence system. ACKNOWLEDGEMENTS This study was supported in part by Grants-in-Aid for Scientific Research (Nos 03660307 and 09660320) from the Ministry of Education, Science and Culture, Japan. REFERENCES DEL CACHO E, GALLEGO M, SANZ A, ZAPATA A (1993) Characterization of distal lymphoid nodules in the chicken caecum. Anatomical Record 237, 512 517. ELDRIDGE JH, HAMMOND CJ, MEULBROEG JA, STAAS JK, GILLEY RM, TICE TR (1990) Controlled vaccine release in the gut

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