Gut-associated lymphoid tissue in the large intestine of calves. II. Electron microscopy

Scanning electron microscopy of lymphoid tissue in the large intestine of three germfree calves (age 3, 6, and 7 days) revealed two different units: propria nodules and lymphoglandular complexes (LGC). Propria nodules had lymphoid tissue predominantly in lamina propria and were covered by distinct follicle-associated epithelium which lacked goblet cells; nodules were surrounded by wide crypts, which were also lined by follicle-associated epithelium towards the luminal side. Lymphoglandular complexes had lymphoid follicles in the tunica submucosa; epithelial diverticulae extended through the muscularis mucosae branching into the lymphoid nodule. In centers of lymphoglandular complexes, protrusions of lymphoid tissue were covered with distinct follicle-associated epithelium. By transmission electron microscopy cells compatible with M cells in the small intestine of calves and cells with characteristics of both enteroabsorptive and M cells were found. Follicle-associated epithelium of propria nodules and lymphoglandular complexes differed only in the relative frequency of cell types.     

Gut-associated lymphoid tissue in the large intestine of calves. I. Distribution and histology

Gut-associated lymphoid tissue (GALT) in the large intestine was characterized in 12 calves (10 to 84 days old) obtained at necropsy (7, group A) or healthy animals (5, group B). Patches of mucosal lymphoid follicles were in all calves at ileocecal entrances (ICE), 23-42 cm distal to the ICE in the proximal loop of the ascending colon (proximal colon [PC] patch), and in the terminal rectum. PC patches varied from 8 to 30 cm in length. Solitary lymphoid follicles were found in the cecum of three calves, between the ileocecal entrances and the PC patch in four calves, adjacent to the PC patch in all calves, and in the ampulla recti. GALT occupied 7.8% of the large intestinal wall in animals of group A: 0.6% at the ileocecal entrance, 4.8% in the proximal colon, and 2.4% in the rectum. There were two different types of mucosal lymphoid follicles in group B: propria nodules with lymphoid follicles predominantly in the lamina propria, and lymphoglandular complexes with lymphoid follicles in the submucosa. In three 3-, 6-, and 7-day-old, germfree calves, distinct follicle-associated epithelium covered propria nodules and covering folds in depths of the lymphoglandular complexes; it was characterized by numerous intraepithelial cells and lack of goblet cells.     

M cells in the rectum of calves

The morphology of gut-associated lymphoid tissue and the ultrastructure of overlying lymphoepithelium of newborn and three-week-old conventionally raised calves were compared. In all calves patches of lymphoid nodules were found in the terminal rectum. In newborn calves lymphoid nodules in the submucosa with caps of lymphoid tissue in the lamina propria predominated. In three-week-old calves lymphoglandular complexes were as numerous as lymphoid nodules with caps. Scanning and transmission electron microscopical examination of superficial lymphoepithelium over caps and lymphoepithelium in epithelial diverticula of lymphoglandular complexes revealed groups or single cells morphologically resembling M cells, but with widely varying apical processes. To investigate whether these putative M cells in rectal lymphoepithelium internalise and transport macromolecules across the epithelial barrier, ferritin was injected into the rectum of three-week-old calves. Eighty to 150 minutes after exposure ferritin was detected in cells resembling M cells. Thus these cells ought to be considered as M cells. It may be hypothesised that gut-associated lymphoid tissue with specialised lymphoepithelium in the rectum of calves provides a route for the uptake of antigen.     

The intestinal habitat for organized lymphoid tissues in ruminants; comparative aspects of structure, function and development

Unlike the Peyer's patches of rats and mice, which are considered to be secondary lymphoid organs, the ileal Peyer's patch of sheep is thought to be responsible for the primary generation of B cells, like the bursa of Fabricius of birds. The ileal Peyer's patch of sheep shows prenatal maturation, antigen-independent lymphopoiesis, a rate of lymphocyte production larger than that of the thymus, and involution at a young age. Follicles contain few T cells and have an IgM+, relatively immature B lymphocyte population, as judged by B-cell differentiation markers. The follicle-associated epithelium of the ileal Peyer's patch is of a special type that sheds carbonic anhydrase-rich, 50-nanometer membrane-bounded particles (carbonic anhydrase-reactive particles; CAP) into the intercellular spaces. The CAP filter into the follicle centre and are taken up by lymphocytes. They represent the epithelial (bursa-like) element in an otherwise mesenchymal stroma of reticular cells embedding the follicle lymphocytes. Transepithelial transport of macromolecules, with the formation of multivesicular body-like cytoplasmic vacuoles, appears to be the basis for CAP formation. The jejunal Peyer's patches are devoid of CAP, persist in the adult animal, contain M cells with clusters of B cells in the follicle-associated epithelium, and have many CD4+ lymphocytes in the follicles and in the interfollicular areas. Aggregates of lymphoid follicles in the large intestine resemble the jejunal Peyer's patches with respect to their lymphocyte population and the ileal Peyer's patch with respect to their follicle-associated epithelium.     

Effect of rotavirus and/or Escherichia coli infection on the aggregated lymphoid follicles in the small intestine of neonatal gnotobiotic calves

The effect of rotavirus and/or Escherichia coli infections on the follicle-associated epithelium (FAE or M cells) of the domes of the aggregated lymphoid follicles (ALF, or Peyer's patches) of gnotobiotic calves was evaluated by light, scanning electron, transmission electron, and immunofluorescence microscopies. Calf rotavirus (CRV) infection produced loss of FAE cell microvilli, and virions were observed in cytoplasmic vacuoles of FAE cells, as well as in intercellular spaces between FAE cells and lymphoid cells migrating through the dome epithelium. The CRV particles appeared to have entered the FAE cells by phagocytosis, with no subsequent cytoplasmic replication. Enterotoxigenic E coli (ETEC) induced more severe alterations including marked microvilli loss and ballooning in the FAE cells. There was no adhesion to, or colonization of FAE cells by ETEC, but bacteria were observed free or phagocytized within the dome and the germinal centers of the ALF. There were no ETEC observed in the cytoplasm of FAE cells. The presence of nonenterotoxigenic E coli (NETEC) in the intestine of calves had no effect on the intestinal FAE cells. The addition of NETEC to CRV infections did not enhance or modify in any way the response of FAE cells to the viral infection; however, the combination of CRV + ETEC produced severe necrosis of the FAE cells, and loss of dome epithelium of ALF.     

Uptake of ferritin and Bordetella avium in bronchus-associated lymphoid tissue of turkeys

The uptake of macromolecular and particulate materials in bronchus-associated lymphoid tissue (BALT) in turkeys was examined using transmission electron microscopy. Tracer materials used were live and ultraviolet-killed (UV-killed) Bordetella avium and ferritin. Suspensions of bacteria and ferritin were instilled via intratracheal catheterization and allowed to remain in contact with the respiratory surfaces for 0, 10, 30, 60, 90, and 120 min. Ferritin and B. avium were taken up by both ciliated and non-ciliated cells of the epithelium overlying BALT (BALT epithelium). Ferritin was found in organelles associated with endocytosis (i.e. apical vesicles, endosomes, cytoplasmic vacuoles) and was apparently transported across epithelial cells, since it was also found in intercellular spaces. Bacteria were found in vacuoles within BALT epithelial cells, but not free in intercellular spaces. Some macrophages in BALT epithelium also contained bacteria. No differences were observed between uptake of live and UV-killed bacteria. We conclude that both ciliated and non-ciliated cells of BALT epithelium in turkeys are able to take up macromolecular and particulate materials. Bacteria are also accessible to intraepithelial macrophages, although whether they are taken up directly from the bronchial surface or whether they pass through epithelial cells first could not be determined. This evidence suggests that antigens, including respiratory pathogens, could gain access to cells of the avian immune system by transepithelial passage in BALT.     

Histology, immunohistochemistry and ultrastructure of the equine tubal tonsil

The tubal tonsil of the horse surrounds the pharyngeal opening of the eustachian tube and is lined by pseudostratified columnar ciliated epithelium interspersed with areas of follicle-associated epithelium (FAE) heavily infiltrated by lymphocytes but devoid of goblet and ciliated cells. Scanning and transmission electron microscopy revealed microvillous cells and cells with features characteristic of M cells such as reduced microvilli or depressed bare surface, more numerous mitochondria, small vesicles and lysosomes, as well as vimentin filaments and epitopes specific for GS 1-B4 as previously seen in the nasopharyngeal tonsil. M cells were also identified in areas of respiratory epithelium not associated with lymphoid follicles and appeared to be the nasal mucosal counterparts of recently described intestinal villous M cells in the mouse. The underlying lymphoid tissue of the FAE was generally organized as solitary lymphoid follicles without germinal centres in contrast to the diffuse and large amount of organized lymphoid follicles with germinal centres that characterize the nasopharyngeal tonsil. CD8+ T and B-lymphocytes were much fewer than in the nasopharyngeal tonsil. High endothelial venules were mainly oriented towards the parafollicular area and contained much fewer endothelial pores and vesiculo-vacuolar organelles. Finally, scattered small clusters of mucus acini and striated muscles were other features that differentiated the tubal and nasopharyngeal tonsils.     

野生黄鼬消化管组织结构的观察

通过组织学观察,探讨野生黄鼬消化管的组织结构特点.将8只野生黄鼬经乙醚麻醉后处死,解剖取食管、胃、小肠、大肠,制作石蜡切片,观察其组织结构.结果显示野生黄鼬食管的黏膜为复层扁平上皮,食管腺发达,肌层以骨骼肌为主.胃贲门部有发达的皱襞和贲门腺;胃底腺有大量的主细胞和壁细胞;胃大弯部的腺体以壁细胞为主,仅有少量主细胞;胃幽门部有发达的幽门腺和大量壁细胞.十二指肠黏膜层有小肠腺,内有潘氏细胞存在,黏膜下层含有十二指肠腺;空肠可见孤立淋巴小结、弥散淋巴组织及集合淋巴小结.结肠无皱襞和肠绒毛,大肠腺排列紧密,其中杯状细胞特别多;直肠固有膜内有发达的大肠腺.所以野生黄鼬消化管的特点是胃各部胃腺发达,壁细胞特别多.     

Differentiation of epithelial cells to M cells in response to bacterial colonization on the follicle-associated epithelium of Peyer's patch in rat small intestine

To clarify the relationship between M cells and intestinal microflora, histoplanimetrical investigation into the bacterial colonization and the differentiation to M cells was carried out in rat Peyer's patch under physiological conditions. The follicle-associated epithelium (FAE), except for the narrow area of apical region, was closely covered with both neighboring intestinal villi and a thick mucous layer, the latter of which also filled the intervillous spaces as well as the space between the FAE and the neighboring intestinal villi. Indigenous bacteria adhered almost constantly to the narrow areas of apical regions of both intestinal villi and the FAE. Bacterial colonies were occasionally located on the basal to middle region of FAE, where M cells also appeared, forming large pockets. When bacterial colonies were located on the basal to middle region of FAE, bacteria with the same morphological characteristics also proliferated in the intervillous spaces neighboring the Peyer's patch. In cases with no bacterial colonies on the basal to middle region of FAE, however, M cells were rare in the FAE. Histoplanimetrical analysis showed the similar distribution pattern of bacterial colonies on the FAE and M cells in the FAE. M cells ultrastructurally engulfed indigenous bacteria, which were then transported to the pockets. These results suggest that indigenous bacterial colonization on the FAE stimulates the differentiation of M cells in the FAE under physiological conditions. The uptake of bacteria by M cells might contribute the regulation of the development of indigenous bacterial colonies in the small intestine.     

Ultrastructural study on the differentiation and the fate of M cells in follicle-associated epithelium of rat Peyer's patch

The differentiation process of immature microvillous epithelial cells to M cells and the fate of M cells in the follicle-associated epithelium (FAE) of the mucosa-associated lymphoid tissues are still unclear. In this study, the differentiation process and the fate of M cells were clarified in rat Peyer's patches under a transmission electron microscope. Almost all immature epithelial cells were found to possess long, slender microvilli, which gradually shortened, thickened and dispersed as the immature epithelial cells migrated away from the crypt orifices. These morphological changes started in the centers and moved to the peripheries of the apical surfaces of epithelial cells, accompanied by the protrusion of apical cytoplasm out of the terminal web. During these changes, the bundles of microfilaments of microvilli never shortened, and both small vesicles in the apical cytoplasm and tiny invaginations of the apical membranes were found. The intraepithelial migrating cells gradually accumulated to form typical intraepithelial pockets. In all FAE, there was no morphological sign of cell death in M cells. The rearrangement of microfilament bundles, the reconstruction of microvilli and the disappearance of pockets resulted in the transformation of M cells into microvillous epithelial cells. These serial ultrastructural changes suggest that M cells are a temporal and transitional cell type caused by the active engulfment of luminal substances and that when the engulfment ceases, the M cells transform into mature microvillous epithelial cells.     

Functional morphology of the large intestinal mucosa of horses (Equus przewalskii f. caballus) with special regard to the epithelium

The mucous membrane of the caecum and colon ascendens of adult horses was first studied using light and transmission electron microscopy. In the surface epithelium there was an inconspicuous constellation of organelles, otherwise there was a lot of mitochondria as a source of energy for absorptive performances. Moreover, enlarged intercellular spaces exist as an indication of an increased uptake of water and electrolytes. In the basal region of Lieberkühn's crypts there were single enteroendocrine cells and numerous granules in the apical epithelial cytoplasm. The functional meaning of these granules is contrarily discussed in the literature. There was no epithelial activity of alkaline phosphatase. Against that a small positive reaction of adenosine triphosphatase was observed at the lateral plasmalemata of the epithelial cells. The sense of these findings with regard to the transport of substances in the equine large intestine is discussed.     

Follicle associated epithelium of the gut associated lymphoid tissue of cattle

The morphology of the gut-associated lymphoid tissue of the small and large intestine in three gnotobiotic calves was examined by scanning and transmission electron microscopy, and the distribution of specialized membranous cells present in the follicle associated epithelium was defined. Isolated follicles remaining in the ileum of a cow after involution of the continuous Peyer's patch were examined by scanning electron microscopy. The presence of membrane-bound particles, reported to be exclusively associated with the continuous Peyer's patch, was investigated in other gut-associated tissue of the small and large intestine of the calf. The presence of two types of follicle associated epithelium in the small intestine of the calf was confirmed, and the follicle associated epithelium of the large intestine proved to be a homogeneous population of specialized membranous cells, similar to that of the continuous Peyer's patch of the small intestine. In the discrete Peyer's patches, some specialized membranous cells were completely hidden by adjacent enterocytes and could only be identified by cytoplasmic extensions into the intestinal lumen. In the proximal part of the continuous Peyer's patch, a transitional zone was detected where the follicle associated epithelium of some doomed villi was composed of a homogeneous population of specialized membranous cells, while the epithelium covering other doomed villi consisted of a mixture of absorptive and specialized membranous cells, usually only found in the discrete Peyer's patches. Membrane-bound particles were observed associated with gut-associated lymphoid tissue in the small and large intestine.     

Investigation of bovine gut associated lymphoid tissue (GALT) using monoclonal antibodies against bovine lymphocytes

Gut associated lymphoid tissue of the small and large intestine of calves and cows has been compared morphologically and quantitatively using monoclonal antibodies to bovine lymphocytes. B cells were significantly decreased in the ileum of the cow compared to the calf. Significantly increased numbers of T cells were present in cell suspensions of all lymphoid areas of the cow compared to the calf. T lymphocyte subsets were quantified into cryostat sections of lymphoid tissues expressing BoT4, and BoT8 antigens demonstrated increased numbers in follicular and dome areas of the discrete Peyer's patches of the small and large intestine of the cow. BoT4+, BoT8+, and the non-BoT4/BoT8+ T cell subsets were increased in the mucosa of the cow as compared to the calf. Similarities in structure and lymphocyte composition of the discrete Peyer's patches of the small intestine, cecum and colon and isolated single follicles in the large intestine suggest similar functional properties.     

Structure of the lining epithelium of the cauda epididymis of the golden hamster

The ductus epididymis has roles in the maturation and storage of spermatozoa. The main function of the cauda epididymis is the storage of spermatozoa; however, this region exerts other morphophysiological roles. So, this study was aimed at investigating structural features of the cauda epididymis epithelium, which could indicate roles other than the storage. The relative percentages of the cell types in the epithelium were 74.9, 6.9, 12.5 and 5.6% of principal, clear, basal and halo cells respectively. Large intercellular spaces were seen among the lateral plasmatic membranes of adjacent principal cells or among these cells and others cell types. These spaces were found to be filled with multivesicular bodies, myelin figures, scrolls and debris of membranes or flocculent dense material. Clear cells had the cytoplasms filled with lysosomes (¾ of basal cytoplasm), and vacuoles and vesicles (¼ of apical cytoplasm). The observations allowed us to infer that clear cells could act in the process of endocytosis and also in water transfer from the lumen to the interstitium through the epithelium compartment. Moreover, transcytosis may occur at the cauda epididymis of Golden hamster.     

Uptake of Mycobacterium avium subsp. paratuberculosis through the distal small intestinal mucosa in goats: an ultrastructural study

Various pathogens gain access to the intestinal wall via specialized cells, the M cells, found among the follicle-associated epithelial cells overlying the domes of the Peyer's patches. The present study was undertaken to examine the uptake of live Mycobacterium avium subsp. paratuberculosis in the distal small intestine of goat kids. Following laparotomy, distal small intestinal segments of five goats were ligated and injected with bacterial suspension. After 1 hour, the intestinal segments were excised and fixed for light and electron microscopic studies. M. a. paratuberculosis organisms were observed by transmission electron microscopy at locations in the intestinal wall, suggesting transcellular transportation through the M cells. The organisms were present both in the cytoplasm of the M cells and in the cytoplasm of intraepithelial leukocytes found in M-cell pockets. Intercellular bacteria between M cells were occasionally seen. Bacteria were not observed in association with the absorptive epithelium. This study indicates that in goat kids, M. a. paratuberculosis enters the intestinal wall primarily through the M cells in the follicle-associated epithelium of the Peyer's patches.     

Large intestinal epithelium and short-chain fatty acids. Expected, unexpected, unexplained findings

Short-chain fatty acids (SCFA) are present in hindgut contents in high concentrations. SCFA are generated and also absorbed rapidly in the large intestine. Absorption results from diffusion of the undissociated and lipid soluble form or in exchange for bicarbonate. The controversial concepts concerning the extent of diffusion or the exchange for bicarbonate are partly due to contradictory findings and unequal mechanisms in different species and in different segments of the large intestine as well as in the different methods used. An almost neutral pH microclimate at the luminal surface is of importance for absorption of SCFA. The apical membranes of the epithelial cells in caecum and in proximal colon of guinea pigs are an substantial barrier for the diffusion of SCFA. After withdrawal of butyrate for only one hour from the perfusion or incubation solutions a massive apoptosis had been observed during the in situ perfusion of segments of guinea pig large intestine and also in in vitro studies with isolated epithelia in Ussing-chambers. In vitro apoptotic bodies and cells are emitted at the epithelial surface. However, in vivo induced by butyrate deprivation resident macrophages were tightly clustered below the surface epithelium. Aided by cytoplasmatic projections these macrophages phagocytose and transport apoptotic material from the epithelial intercellular spaces into their bodies. Obviously macrophages can be overloaded by this massive apoptosis, and some of the undigested apoptotic fragments are emitted into the lamina propria. Formation of a colitis ulcerosa may originate from these released undigested apoptotic bodies. Furthermore hints indicate aetiological interrelations between deprivation of butyrate and colon cancer. Butyrate-paradox denotes the contrarian effects on apoptosis and cell proliferation after addition or deprivation of butyrate in cultures of large intestinal tumour-cell-lines in comparison with the healthy, intact epithelium.     

Bovine ileal dome lymphoepithelial cells: endocytosis and transport of Brucella abortus strain 19

Ligated ileal loops of calves were inoculated with Brucella abortus and examined at 2, 4, 6, 10, and 24 hours post-inoculation. B. abortus was identified by light and electron microscopy using immunoperoxidase and antibody-coated colloidal gold techniques. B. abortus was detected in vesicles, phagolysosomes, and large vacuoles of lymphoepithelial cells. Numbers of intracellular bacteria decreased with time after inoculation. B. abortus was also seen between and below lymphoepithelial cells and free in the dome interstitium and intestinal lymph vessels. Neutrophils and macrophages in both epithelium and lamina propria contained intact or degraded bacteria within phagosomes, phagolysosomes, and multivesicular bodies. These studies showed that (1) transepithelial migration of B. abortus occurred principally by dome lymphoepithelial cell endocytosis and transport, and (2) B. abortus was degraded by macrophages and neutrophils of the gut-associated lymphoid tissue.     

Pathology of proliferative haemorrhagic enteropathy in pigs.

Examination of the small intestine of pigs with proliferative haemorrhagic enteropathy showed changes consistent with defects in vascular permeability. Early in the disease there were many eosinophils and distension of lacteals and intercellular spaces with proteinaceous material. Later the predominant features were red blood cells and exudate in tissue spaces. This was most severe and extensive at the tips of villi which were covered by a cast of cells and fibrinous exudate. Adenomatous intestinal mucosal cells contained organisms that were free within the apical cytoplasm and were morphologically identical with those seen in the related disease, porcine intestinal adenomatosis. Also these bacteria were seen free in the subepithelial mucosal area, in blood vessels and within membrane-bound vesicles in phagocytic cells in the mucosa and its blood vessels. Mast cells were prominent in some areas as were thrombosed vessels.     

An immunohistochemical study of the organization of ganglia and nerve fibres in the mucosa of the porcine intestine

In order to elucidate the organization of the enteric nervous system in the mucous plexus, wholemounts from six intestinal regions in six pigs were studied by vasoactive intestinal peptide, substance P, nitric oxide synthase and neurofilament proteins immunohistochemistry. The mucous plexus of both large and small intestine contained ganglia and isolated neurons. They were many and comparably larger in the caecum and colon, few in the ileum, and fewer and smaller in the jejunum. The mucous plexus was subdivided into the lamina muscularis mucosae and lamina proprial subplexuses, and based on location the latter was subdivided further in order to clarify their variations with respect to the amount, sizes and shapes of ganglia and neurons, sizes and orientation of nerve strands and immunoreactivities. Ganglia were situated at different topographical levels in the lamina muscularis mucosae subplexus, outer proprial and interglandular proprial meshworks in the lamina proprial subplexus with the majority of ganglia occurring in the outer proprial meshwork. The mucous plexus in the intestine of the pig is thus a ganglionated plexus showing marked segmental variation in the amount of intramucosal ganglia and isolated nerve cells. These new observations, calls for a re-examination of the mucous plexus to elucidate the regulatory mechanisms of importance in mucosal functions and consideration of the mucous plexus in the intestine of the pig to be one of the major ganglionated plexuses.     

Differences in lectin-binding properties between the common mucosal epithelium and follicle-associated epithelium in the rabbit small intestine

Differences in sugar distribution between the villous epithelium and follicle-associated epithelium (FAE) were compared using lectins in the rabbit small intestine. In every portion, villous columnar epithelial cells primarily exhibited a positive reaction to the GalNAc, GlcNAc, galactose, and oligosaccharide. In the ileal Peyer's patch (PP), whereas microvillous epithelial cells exhibited positive reactions, M cells tended to be negative. The villous epithelial reaction to the fucose group was negative, but M cells and microvillous epithelial cells showed a positive to the fucose. No epithelium had a positive reaction to the mannose and glucose. The variety of lectin-binding properties of villous epithelial cells and M cells may reflect specificity for the recognizing luminal substances such as antigenic molecules and bacterial elements.