Lymphoglandular Complexes in the Large Intestine of the Dog

J. Anat. (1972), 113, 2, pp. 169-178 169 With 4 figures Printed in Great Britain

Lymphoglandular complexes in the large intestine of the dog

A. M. ATKINS AND G. C. SCHOFIELD Department of Anatomy, Monash University, Clayton, Victoria, Australia (Accepted 18 August 1972)

INTRODUCTION In the human intestine there are many lymphoid cell aggregates located in the submucous layer. Some extend to a varying degree into the adjacent mucous membrane, and large lymphoid aggregates, for example Peyer's patches, often envelop mucosal glands and extend as far as surface epithelium (Patzelt, 1936). Similar relationships have been reported in many other mammalian species (Oppel, 1897). An even more intimate association of lymphoid tissue and intestinal epithelium occurs in some species in which intestinal glands in localized regions penetrate the muscularis mucosae and expand and branch within submucosal lymphoid nodules (Hebel, 1960); in the Australian echidna such complexes were referred to by Scho- field & Cahill (1969) as lymphoepithelial glands. We report here the presence of similar complexes in the intestine of the dog, a species well suited for immunohistochemical, developmental, and morphologic investigations.

MATERIALS AND METHODS Studies were carried out on intestinal tissues obtained under anaesthesia from 14 healthy adult dogs ofvarious strains and ages. Specimens were taken from the caecum, and at intervals throughout the colon, rectum and anal canal. Specimens were also taken from corresponding regions of the large intestine of fetal and neonatal dogs. Additional material was obtained from eight fetuses, two from each of four litters having an estimated gestation period ranging between 5 and 8 weeks, four newborn pups including two from each of two litters, and two pups each of 4 weeks of age. The mucosal surface of unfixed specimens was examined using a dissecting micro- scope. For light microscopy, specimens of intestine were fixed in cold 10 % neutral formalin, in Hollande's solution, or in Bouin's solution; alternatively they were freeze-dried. Other specimens were fixed in 4 % glutaraldehyde or in 4 % glutaraldehyde in 10 % acrolein, and were examined by light microscopy after embedding in glycol methacrylate (Feder & O'Brien, 1968). Several histochemical staining reactions were performed on tissue sections using routine techniques, and sections of freeze-dried tissues were also examined for the presence of amine fluorophores, using the formaldehyde-induced fluorescence histochemical technique (Falck & Owman, 1965). For ultrastructural studies small blocks were fixed in 4 % phosphate- buffered glutaraldehyde (Sabatini, Bensch & Barrnett, 1963), and postfixed in 2-5 % osmium tetroxide in potassium dichromate (Dalton, 1955). 170 A. M. ATKINS AND G. C. SCHOFIELD Immunohistochemical studies involved tissues from adult dogs using rabbit antisera directed against canine serum IgG, against canine serum IgM, and against a fraction of canine colostrum containing an immunoglobulin identified as IgA. A fraction of canine serum containing IgM, and contaminated by traces of non- immunoglobulin proteins, was prepared according to Reynolds & Johnson (1970). A cruder preparation of IgG was separated from canine serum using diethylamino- ethyl-cellulose (DEAE-cellulose) (Reif, 1969). An IgA-rich fraction of colostrum was also obtained using essentially the method of Reynolds & Johnson (1970). The colostrum used was obtained from several dogs in the first 2 days following parturition; the whey was separated, filtered, dialyzed against borate-buffered saline (pH 8 0), and chromatographed on a column of Sephadex G-200 in 01M phosphate buffer (pH 7 0). Components of a concentrate of the appropriate fractions were then further separated on Whatman microgranular DEAE-cellulose, DE-52, by elution with a continuous salt gradient. The development of rabbit immune sera, their conjugation with fluorescein, and the immunofluorescence staining oftest and control preparations employed techniques described previously (Atkins, Schofield & Reeders, 1971). Immunoelectrophoresis of the immunoglobulin antigens and corresponding antisera was performed by modification of the technique of Scheidegger (1955). Rabbit antisera directed against the IgA-rich colostral fraction were shown to be active also against IgM. These antisera were therefore absorbed with canine serum IgM prior to being used for immunofluorescence staining.

OBSERVATIONS Macroscopic appearances In the caecum of each adult dog studied, structures subsequently identified histo- logically as the openings of lymphoglandular complexes were seen on macroscopic examination of the mucosal surface. These fine openings were situated at the centre of smooth rounded elevations approximately 3 mm in diameter, which were distributed uniformly throughout the caecum, both on and between irregularly disposed mucosal folds, and averaged approximately three per cm2 of mucosal surface. Similar structures were present in smaller numbers in the colon adjacent to the caecal opening, but were not seen either macroscopically or in histological preparations of the remainder of the large intestine. Light microscopy in adult tissues In adult dogs, submucosal lymphoid nodules invaginated by submucosal extensions of overlying intestinal glands were identified in the caecum and proximal colon and were designated lymphoglandular complexes (Fig. 1). Mucosal lymphoid tissue and submucosal lymphoid nodules not associated with invaginating glands were also seen in each of the major topographical subdivisions of the large intestine, including the caecum and the immediately adjacent part of the colon. Submucosal lymphoid nodules which formed part of lymphoglandular complexes were rounded or oval in section and their greatest diameters ranged between 0-4 and 1 0 cm. The nodules were closely applied to the deep aspect of the muscularis mucosae and they seldom occupied the whole thickness of the submucosa. They were invested Lymphoglandular complexes in large intestine 171

1 Fig. 1. Section of a lymphoglandular complex in the caecum of an adult dog. Intestinal glands opening into a depression on the surface of the mucous membrane and extending into a submucosal lymph nodule can be seen. The position of a deficiency in the muscularis mucosae is indi- cated by arrows. PAS. x 20. by. a condensation of submucosal connective tissue containing small vessels which entered the nodules. The mucous membrane overlying the centre of each lymphoglandular complex presented either a saucer-shaped depression or a pit, in each case usually occupying the full thickness of the mucous membrane and invariably lined by epithelium. Many of the columnar epithelial cells lining the depression contained 172 A. M. ATKINS AND G. C. SCHOFIELD supranuclear granules which were PAS-reactive even after diastase digestion. From the depths of the pit, glands resembling mucosal glands extended into the submucosal lymphoid nodule through a localized deficiency in the muscularis mucosae. The epithelial cells lining the submucosal glandular extensions ranged from squamous to columnar in outline and contained few PAS-reactive granules. Goblet cells were absent at the fundus and scanty elsewhere in the submucosal glands. Many lymphoid cells were seen within the epithelium of the complexes. Whatever their location in the large intestine, submucosal lymphoid aggregates were homogeneous in appearance. There was no evidence of division into cortical and medullary areas, and germinal centres were also absent. Lymphocytes were the predominant cells present, though large pyroninophilic cells, usually more centrally placed but not obviously associated with the epithelium of lymphoglandular complexes, and phagocytes, identified by their acidophilic cytoplasm or PAS-reactive granules, were also identified. The formaldehyde-induced fluorescence technique revealed a few fine nerve fibres in the mucosa and submucosa, and particularly in relation to the muscle layers. Fluorescent nerve fibres were not seen in or near simple submucosal lymphoid nodules or in relation to lymphoglandular complexes. Cells containing an amine fluorophore and identified as enterochromaffin cells were seen in glands of the mucous membrane, but were not present in the submucosal glandular component of lymphoglandular complexes. Granules which emitted a non-specific yellow-orange autofluorescence were also seen in a few cells within the lymphoid nodules. Their fluorescence was similar to that of lipofuscins reported in other canine tissues (Edwards, Atkins & Schofield, 1972). Light microscopy in prenatal and young animals Histological examination of serial sections of tissues obtained from fetal animals failed to detect either lymphoglandular complexes or lymphoid aggregates in any of the parts of the large intestine studied. In the caecum and in other areas of the large intestine of newborn pups, small aggregates of lymphoid cells were seen in the submucosa beneath the relatively undifferentiated mucous membrane. In the caecum of pups aged 4 weeks, intestinal glands which perforated the muscularis mucosae and entered the submucosal layer to varying depths were occasionally seen. The submucosal glandular extensions were not associated with any striking modification of the mucosal surface and, though different in location, were similar in appearance to adjacent mucosal glands. However, submucosal connective tissues adjacent to the glandular extensions contained small aggregations of lymphoid cells which were not observed in other areas of the submucosa. Immunohistochemical studies When fluorescein-conjugated rabbit antiserum directed against canine colostral IgA was used, immunofluorescence staining was seen in adult tissues in cells of the lamina propria identified as plasma cells, in columnar epithelial cells of mucosal glands (Fig. 2) (but not in the submucosal glands of lymphoglandular complexes), and in cells of submucosal lymphoid nodules. IgA-containing plasma cells were distributed regularly throughout the lamina propria of the caecum and colon, including the region of lymphoglandular complexes; most of the cells showed homo- Lymphoglandular complexes in large intestine 173

Fig. 2. Fluorescein-conjugated antiserum directed against canine colostral IgA has been applied to a methanol-fixed section of dog caecum. Fluorescence in the epithelium is located mainly in the apical cytoplasm of columnar cells lining mucosal glands and is absent from the mucinogen mass of goblet cells. Numerous plasma cells (P) throughout the lamina propria are also stained. In addition, groups ofcells with intensely autofluorescent inclusions (A) can be seen in the lamina propria. x 100. geneous cytoplasmic staining but some contained discrete fluorescent granules. In most of the columnar epithelial cells immunofluorescence staining was more pronounced in the apical region. It was not possible to distinguish which category of cell in lymphoid aggregates showed immunofluorescence. After staining with either fluorescein-conjugated rabbit antiserum directed against canine serum IgG, or with corresponding antiserum directed against serum IgM, cytoplasmic fluorescence was seen in cells in the lamina propria identified as plasma cells and in a few lymphoid cells in some of the submucosal lymphoid nodules. Fluorescence staining was not seen in epithelial cell cytoplasm in any part of the large intestine with the use of either antisera. The fluorescent lymphoid cells seen in tissue preparations stained by either antiserum were approximately equal in number; both types of cell were evenly distributed. Lymphoid cells and plasma cells containing either IgM or IgG were more numerous than IgA-containing cells in submucosal lymphoid aggregates, and were generally less numerous in the lamina propria. 174 A. M. ATKINS AND G. C. SCHOFIELD .pq0

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ANA II3 176 A. M. ATKINS AND G. C. SCHOFIELD Control staining of serial sections carried out for each antiserum used failed to show fluorescence in any cell type. Ultrastructural observations on lymphoglandular complexes Lining the mucosal part of each lymphoglandular complex were columnar and goblet cells which were indistinguishable from corresponding cells lining mucosal glands nearby. The columnar cells contained electron-dense granules resembling those found in man, sheep, and pig, and identified as IgA-containing secretory inclusions. They were found in relation to the Golgi complex, and, more commonly, near the microvillous border. Secretory inclusions were less evident in the epithelium of the submucosal part of lymphoglandular complexes (Fig. 3). The epithelium throughout lymphoglandular complexe3 contained numerous lymphoid cells, ranging from relatively undifferentiated lymphoblasts to small lymphocytes. Cells, designated plasmablasts and plasma cells by virtue of their nuclear form and their content of rough endoplasmic reticulum and polyribosomes, were also seen within the epithelium. The lymphoid cells were usually arranged in nests occupying spaces between the epithelial cells and, excepting for their location, were similar in appearance to cells seen in the adjacent lymphoid tissues (Fig. 4). Both normal and degenerated lymphoid cells were also present in the lumen of lymphoglandular complexes, particularly at the fundus of submucosal glandular extensions. The lymphoglandular complexes contained a heterogeneous collection of lymphoid cell types, many of which could not be identified with certainty on the basis of their ultrastructural appearance alone. Small lymphocytes were predominant. Lymphoblast forms, rich in polyribosomes and lacking an endoplasmic reticulum, plasmablasts with a ribosome-rich cytoplasm but small amounts of granular endoplasmic reticulum, and typical plasma cells were seen commonly throughout the nodules. DISCUSSION Bonnet (1880) originally described solitary lymphoid follicles in the caecum of the dog but did not refer to the presence of epithelial elements within them. The lymphoglandular complexes described here comprise branching intestinal glands which penetrate submucosal lymphoid nodules. A similar association of epithelial and lymphoid elements in the large intestine has been reported in several species including the dog (Hebel, 1960). Schofield & Cahill (1969) referred to the similarity between lymphoepithelial glands in the adult echidna and the avian bursa of Fabri- cius, and suggested that the lymphoepithelial glands represent bursal equivalents which, in later life, adopt a role as secondary lymphoid organs. Developmental studies on the echidna, particularly in early post-hatching stages, cannot readily be carried out, and studies on other species are necessary to establish the times of appearance of lymphoepithelial complexes. In respect of their form and staining reactions, including immunohistochemical reactions, the columnar epithelial cells of mucosal glands in the large intestine of the dog are similar to those previously reported in corresponding areas in monkey, sheep, pig and man (Schofield, 1970; Schofield & Atkins, 1970; Atkins, Schofield & Reeders, 1971). The cells thus appear to represent sites at which IgA, presumably Lymphoglandular complexes in large intestine 177 originating from plasma cells in the lamina propria, is accumulated prior to its discharge into the lumen. However, the epithelium of lymphoglandular complexes undergoes minor changes in appearance as it invades submucosal lymphoid aggregates. Goblet cells are here less numerous and evidence of secretory activity of columnar epithelial cells is less pronounced than elsewhere in the mucous membrane. Other immunohistochemical studies have shown that IgA-containing plasma cells are more numerous in the mucous membrane near Peyer's patches (Crabbe et al. 1970; Atkins et al. 1971). In the large intestine of the dog, however, it would appear that lymphoglandular complexes do not make a significant contribution to the immunoglobulin component of intestinal secretions. Observations on the appearance, development and regression or experimental removal of gut-associated lymphoid aggregates of rabbits have led to their classifica- tion as primary lymphoid organs with a role equivalent to that of the avian bursa of Fabricius (Cooper, et al. 1966). This view has not been supported by the results of studies on lymphocyte migration patterns (Gowans & Knight, 1964) or by findings from lymphocyte repopulation experiments using irradiated mice (Evans, Ogden, Ford & Micklem, 1967). There is indeed a resemblance in basic morphology between the avian bursa of Fabricius and the lymphoglandular complexes in the large intestine of adult dogs but the developmental history of the complexes makes it unlikely that they function as primary lymphoid organs. For example, the lymphoglandular complexes are not fully assembled until several weeks after birth, that is, at a time when secondary lymphoid tissue is well developed and when humoral immune capacity is known already to have been established (Jacoby, Dennis & Griesemer, 1969). The presence of plasma cells within the complexes of adult dogs is compatible only with their having a role at this stage as secondary lymphoid organs. More detailed studies will be necessary to explore any difference in immuno- logical role between lymphoid aggregates which are part of lymphoglandular complexes and those which are not. Any such differences are likely to depend on the presence of extensions of the intestinal lumen, and thus relatively free access of intestinal contents, including micro-organisms, into the submucosal lymphoid tissue of the complexes.

SUMMARY Lymphoglandular complexes in the large intestine of the dog have been studied using histochemical, immunohistochemical and ultrastructural techniques. The complexes are found only in the caecum and immediately adjacent part of the colon and they comprise submucosal extensions of intestinal glands branching within submucosal lymphoid nodules. The structure and development of the complexes indicate that their lymphoid component has a role as a secondary lymphoid organ. In the dog it thus appears likely that the development of humoral capacity is not associated with, or dependent on, the presence of lymphoglandular complexes. We are indebted to Mr A. Bond of the Baker Medical Research Institute, Mel- bourne, for assistance in obtaining tissue specimens, to Mr P. R. McKinnon and Miss S. Eckert for technical assistance, and to Mr J. S. Simmons, F.R.P.S., for the photographic reproductions. 12-2 178 A. M. ATKINS AND G. C. SCHOFIELD This work was supported by a grant from the National Health and Medical Research Council of Australia. REFERENCES ATKINS, A. M., SCHOFIELD, G. C. & REEDERS, T. (1971). Studies on the structure and distribution of immunoglobulin A-containing cells in the gut of the pig. Journal of Anatomy 109, 385-395. BONNET, R. (1880). Die solitaren Follikel im Blinddarm des Hundes. Zeitschrift fur Tiermedizin 2, 307-309. COOPER, M. D., PEREY, D. Y., MCKNEALLY, M. F., GABRIELSEN, A. E., SUTHERLAND, D. E. R. & GOOD, R. A. (1966). A mammalian equivalent of the avian bursa of Fabricius. Lancet i, 1388-1391. CRABBE, P. A., NASH, D. R., BAZIN, H., EYSSEN, H. & HEREMANS, J. F. (1970). Immunohistochemical observations on lymphoid tissues from conventional and germ-free mice. Laboratory Investigation 22, 448-457. DALTON, A. J. (1955). A chrome-osmium fixative for electron microscopy. Anatomical Record 121, 281. EDWARDS, G. M. H., ATKINS, A. M. & SCHOFIELD, G. C. (1972). Studies on 'argentaffin' inclusions in the submandibular gland of the dog. Journal of Anatomy 110, 494. EVANS, E. P., OGDEN, D. A., FORD, C. E. & MICKLEM, H. S. (1967). Repopulation of Peyer's patches in mice. Nature, London 216, 36-38. FALCK, B. & OWMAN, C. (1965). A detailed methodological description of the fluorescence method for the cellular demonstration of biogenic amines. Acta Universitatis lundensis. Section II 7, 3-23. FEDER, N. & O'BRIEN, T. P. (1968). Plant microtechnique: some principles and new methods. American Journal of Botany 55, 123-142. GOWANS, J. L. & KNIGHT, E. J. (1964). The route of re-circulation of lymphocytes in the rat. Proceedings of the Royal Society, Series B 159, 257-282. HEBEL, V. R. (1960). Untersuchungen uber das Vorkommen von lymphatischen Darmkrypten in der Tunica submucosa des Darmes von Schwein, Rind, Schaf, Hund und Katze. Anatomische Anzeiger 109, 7-27. JACOBY, R. O., DENNIS, R. A. & GRIESEMER, R. A. (1969). Development of immunity in fetal dogs: humoral responses. American Journal of Veterinary Research 30, 1503-1510. OPPEL, A. (1897). Lehrbuch der vergleichenden mikroskopischen Anatomie der Wirbeltiere, Zweiter Teil, II, pp. 409-447. Jena. PATZELT,V. (1936). Die Follikel,Peyeischen Platten und tonsillenartigen Organe des Darmes.InHandbuch der mikroskopischen Anatomie des Menschen: Verdauungsapparat, vol. 3, pp. 214-232. Berlin: Springer. REIF, A. E. (1969). Batch preparation of rabbit yG globulin with DEAE-cellulose. Immunochemistry 6, 723-731. REYNOLDS, H. Y. & JOHNSON, J. S. (1970). Canine immunoglobulins. III. Distribution of immunoglobulins in colostrum and isolation of secretory IgA and 7Sy,. Journal of Immunology 104, 1000-1008. SABATINI, D. D., BENSCH, K. & BARRNETT, R. J. (1963). Cytochemistry and electron microscopy. The preservation of cellular ultrastructure and enzymatic activity by aldehyde fixation. Journal of Cell Biology 17, 19-58. SCHEIDEGGER, J. J. (1955). Une micro-methode de l'immuno-e1ectrophorese. International Archives of Allergy and Applied Immunology 7, 103-1 10. SCHOFIELD, G. C. & CAHILL, R. N. P. (1969). Intestinal and cloacal lymphoepithelial glands in the Australian echidna: a possible homologue of the bursa of Fabricius. Journal ofAnatomy 105, 447-456. SCHOFIELD, G. C. (1970). Columnar cells with secretory granules in the large intestine of the macaque (Cynamolgus irus). Journal of Anatomy 106, 1-14. SCHOFIELD, G. C. & ATKINS, A. M. (1970). Secretory immunoglobulin in columnar epithelial cells of the large intestine. Journal of Anatomy 107, 491-504.