Reduced apoptosis in sheep ileal Peyer's patch is associated with low levels of follicle centre carbonic anhydrase reactivity

Apoptosis in lymphoid follicles of the ileal Peyer's patch (IPP) in 21 sheep of two different age groups was visualized by the TdT-mediated dUTP nick end-labelling (TUNEL) method, and quantified using computer-assisted image analysis. The IPP follicle carbonic anhydrase (CA) reactivity was evaluated in the same samples. No significant differences with respect to apoptosis and CA reactivity were found between sheep aged 5 and 11 months. Individual variation in apoptotic activity correlated with the follicle centre CA reactivity. The group of animals found to have predominantly atypical ileal lymphoid follicles (more than 80% of total number of follicles) with features resembling jejunal Peyer's patch follicles, had lower number of apoptotic cells and reduced CA reactivity compared to the rest of the animals. The differences in CA reactivity in the follicle centres probably represent a variation in the presence of CA rich approximately 50 nm membrane-bounded particles known to be a feature of the sheep IPP. The present results suggest that the particles are involved in the modulation of the lymphocyte proliferation of the IPP follicles.     

Uptake of colostral leukocytes in the intestinal tract of newborn calves

The role of colostral immunoglobulins for the protection of newborn calves has been studied extensively, but little is known about the importance of colostral leukocytes. To study the uptake of colostral leukocytes in the intestine of calves and to determine preferential sites for this uptake, FITC-labelled colostral cells derived from the respective dams were injected into intestinal loops with/without Peyer's patches of three male Holstein Frisian calves about 5h post natum. In adjacent loops, PBS was injected as control. Loops were excised after an exposure of 1.5-2h. FITC-labelled material and cells were detected by the direct immunoperoxidase method in paraplast sections. Twenty-five consecutive sections were evaluated from each localization. Uptake of labelled material and cells was observed in all three calves in the jejunal Peyer's patch and in two calves in the ileal Peyer's patch as well. In the jejunal Peyer's patch, labelled material and cells were present in epithelium, domes and sinuses around lymphoid follicles, whereas in the ileal Peyer's patch, they were found in the sinuses only. These findings confirm that uptake of colostral leukocytes through the intestinal barrier is possible and that the preferential route of uptake is through follicle-associated epithelium of Peyer's patches.     

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.     

Distribution of lymphocyte subsets in the small intestine lymphoid tissue of 1-month-old lambs

Distribution of lymphocyte subpopulations along the small intestine lymphoid tissue has been examined in 1-month-old lambs using flow cytometric and immunohistochemical techniques. Monoclonal antibodies against CD4, CD8, gamma delta, CD45R and B receptors have been employed in samples from continuous ileal Peyer's patch (IPP), discrete jejunal Peyer's patches (JPP), ileocaecal valve lymphoid tissue (ICVPP), mesenteric lymph node (MLN) and intra-epithelial (IEL) and lamina propria (LPL) lymphocytes. Histological studies were also done. Differences in the lymphocyte distribution have been observed between some of the regions examined, especially between IPP and JPP for most of the markers. A remarkable feature was the existence of morphological and lymphocyte distribution differences between ICVPP and IPP, locations that had been traditionally considered as similar. The antibody against CD45R receptor used in this study, that was supposed to mark B cells and some T cells, detected cell populations located in the dome of the follicles in all the samples, whereas the centre was negative. Lymphocytes positive to the B marker employed were located mainly in the centre, suggesting that both antibodies would mark B cells in different maturation status.     

Ileal Peyer's patches in experimental infections of calves with rotaviruses and enterotoxigenic Escherichia coli: a light and electron microscopic and enzyme histochemical study

The effect of rotaviruses and enterotoxigenic Escherichia coli administered in various sequences to cesarean-derived, colostrum-deprived calves was studied using light and electron microscopy. The structure of the lymphoid tissue in the ileum, the number of mitoses in the crypts, number of intraepithelial lymphocytes, and enzyme histochemistry (alkaline phosphatase, acid phosphatase, succinic dehydrogenase, beta-galactosidase, and leucinaminopeptidase) of the ileal dome epithelium were evaluated. The area of lymphoid follicles in Peyer's patches of the ileum was investigated morphometrically. Monoinfections with either rotavirus or enterotoxigenic E. coli induced a significant increase in lymphoid follicle area, but did not affect dome epithelial cells. Dual infections did not consistently affect the follicle area, but the number of intraepithelial lymphocytes and the mitotic indices exceeded those of comparable monoinfections. Changes in activity of enzymes in the ileal dome epithelial area were minor.     

The sheep and cattle Peyer's patch as a site of B-cell development

In sheep and cattle, the ileal Peyer's patch (PP), which extends one-two meters along the terminal small intestine, is a primary lymphoid organ of B-cell development. B-cell diversity in the ileal PP is thought to develop by combinatorial mechanisms, gene conversion and/or point mutation. These species also have jejunal PP that function more like secondary lymphoid tissues concerned with mucosal immune reactions. These two types of PP differ significantly in their histology, ontogeny and the extent of lymphocyte traffic. The prenatal development of follicles in the PP begins first in the jejunum during the middle of gestation and then in the ileum during late gestation. B-cells proliferate rapidly in the ileal PP follicle; up to five percent of these cells survive while the majority dies by apoptosis, perhaps driven by the influence of environmental antigen and/or self-antigen. The surviving cells migrate from the ileal PP and populate the peripheral B-cell compartment. By adolescence, the ileal PP has involuted but the function of jejunal PP, compatible with a role as secondary lymphoid organ, continues throughout life. In this review, we focus on the development of PP as a site of B-cell repertoire generation, positive and negative B-cell selection, and the differences between ileal PP and jejunal PP.     

Age, scrapie status, PrP genotype and follicular dendritic cells in ovine ileal Peyer's patches

Follicular dendritic cells (FDCs) residing within ileal Peyer's patches (PPs) are of crucial relevance for sheep scrapie early pathogenesis and subsequent scrapie prion neuroinvasion. In this study, ileal PP follicles were significantly more numerous in lambs than in adult Sarda breed sheep, with significant differences being also found in lymphoid follicle area, perimeter and FDC density. Furthermore, PrPd deposition within ileal PPs and host's PrP genotype did not significantly influence these parameters. We conclude that age significantly affects FDC density in ileal PPs from Sarda breed ovines, independently from host's scrapie status and PrP genotype.     

Immunoperoxidase studies of cell mediated immune effector cell populations in early Mycobacterium avium subsp. paratuberculosis infection in sheep

Immunoperoxidase (IPX) labelling for CD4, CD8, TCR-gammadelta, WC1, CD1b, IFN-gamma, CD45R, CD56 and lysozyme was used to investigate changes in cell mediated immune effector cell populations in the intestinal Peyer's patches (PP) and mesenteric lymph nodes of lambs, 2 and 4 months after experimental infection with low doses of sheep strain Mycobacterium avium subsp. paratuberculosis (M. a. paratuberculosis). The organism was cultured from the tissues of each infected lamb, but histological lesions were not present. This infection model was considered to be more representative of natural M. a. paratuberculosis infection than previous studies. Infected sheep had significantly more CD4+ cells in the mucosa, domes and interfollicular areas of the terminal ileum, and in the interfollicular areas of the jejunal Peyer's patch. Infected sheep also had significantly increased numbers of TCR-gammadelta+ cells in the mucosa and interfollicular areas of the jejunal Peyer's patch, and increased numbers of WC1+ cells in the ileal Peyer's patch. These findings are consistent with previous findings in sheep given higher doses of cattle strain M. a. paratuberculosis. Significantly fewer CD1b+ cells were present in the paracortical areas of the mesenteric lymph nodes of infected sheep, and the reduction was greater in sheep infected for 4 months compared to sheep infected for only 2 months. Down-regulation of CD1b expression may be important for the continued survival and multiplication of M. a. paratuberculosis as specific adaptive immunity develops. Across all sheep, jejunal Peyer's patches had higher numbers of CD4+, CD8+, TCR-gammadelta+, WC1+ and CD45R+ cells, and lower numbers of CD56+ fibres compared to ileal Peyer's patches. These findings confirm and extend the peculiarities of the terminal ileal Peyer's patch in the young ruminant, with possible implications for the early establishment of M. a. paratuberculosis infection.     

Characterization and Distribution of B Cells in the Lymphoid Organs of Goats

The distribution of B cells in the lymphoid organs of the goat was studied using a panel of 13 monoclonal antibodies (mAbs) developed against different markers for bovine B cells. Samples of mesenteric lymph nodes, jejunal and ileal Peyer's patches, duodenum, jejunum, ileum, colon, caecum and rectum were taken from four 7-month-old male Murciano-granadina goats using the avidin-biotin-peroxidase (ABC) method on frozen sections as described by Hsu et al. (1981). The mAbs against immunoglobulins (Ig) recognized a large number of cells, particularly in the light zones of the germinative centres of the lymphoid follicles, regardless of the isotype against which they were directed. However, the greatest numbers of B cells in the germinative centres and outer coronas of the lymphoid follicles of the lymph node, spleen and Peyer's patches were recognized by mAbs against the L lambda chain of Ig and against IgM. This was also the case in other locations where B cells were abundant, such as the medulla of the lymph node and the dome of the Peyer's patches. These mAbs recognized not only B lymphocytes but also plasma cells, showing an intracytoplasmatic reaction (numerous in the spleen red pulp and the intestinal lamina propria when mAbs were used against the L lambda chain of the Ig, scarce in the intestinal lamina propria when used against IgM and scarce in spleen red pulp and numerous in the intestinal lamina propria when mAbs against IgA were used). The mAbs BAQ44 A, GC65 A and GB25 A are of interest because, besides marking cells in the B areas where lymphocytes show surface Ig, they give a positive reaction in areas where there are Ig-cells (the dark zone of the germinative centre) and do not immunostain plasma cells. Thus, these mAbs recognize a surface marker which is not an Ig.     

Ultrastructure and alkaline phosphatase activity of the dome epithelium of canine Peyer's patches

The dome epithelium of Peyer's patches from different parts of the intestine of four dogs was examined by scanning and transmission electron-microscopy and by alkaline phosphatase histochemistry on glycol-methacrylate embedded sections. M cells were scattered among more numerous enterocytes in duodenal and jejunal Peyer's patches, but constituted the major cellular component of dome epithelia of the ileal Peyer's patches. Alkaline phosphatase histochemistry demonstrated low enzyme activity in the brush border of M cells, as compared to enterocytes, in the duodenum and jejunum, allowing identification of M cells at the light microscopic level. Alkaline phosphatase activity was too low in ileal enterocytes to permit visualization of M cells. The presence of intrafollicular invaginations of dome epithelium is a consistent finding in duodenal Peyer's patches of the dog and these invaginations were characterized by few M cells, many intraepithelial lymphocytes and strong alkaline phosphatase activity.     

Postnatal development of lymphocyte subsets in different compartments of the small intestine of piglets

In contrast to rodents, all compartments of the porcine small intestine contain lymphoid cells at birth. During the first few days of life maternal antibodies and leukocytes are taken up by the intestinal epithelium. The number of intraepithelial lymphocytes (IEL) increases by a factor of 12 from Day 1 to Day 60, but in germfree pigs only a minor increase is observed. Immediately after birth, low numbers of T cells are present in the small intestinal lamina propria (LP), and many of these express neither CD4 nor CD8 on their surface. This type of subset composition is still present in germfree pigs at an age of 1.5 months. The appearance of IgA+ and IgM+ lymphocytes in the LP differs. Many more of these B cells are found in the LP of the crypts than of the villi. The development of the discrete jejunal Peyer's patches (PP) differs from that of the continuous PP in the terminal ileum. The lymphocyte subset composition shows the most obvious differences between conventional and germfree piglets and between the jejunal and ileal PP at an age of 1.5 months. Several markers, e.g. for antigen-presenting cells, memory T cells, M cells in the domes of PP, have recently become available for the pig. These should now be applied in experiments on pigs in the early postnatal period to study the mechanisms of the development of tolerance and protective immune reactions.     

Quantitative analyses of lymphoid tissue in the spleen, lymph nodes and Peyer's patches in cynomolgus monkeys

To clarify the morphological characteristics of the cynomolgus monkey immune system, we analyzed quantitative data on their lymphoid organs. Spleens, major lymph nodes and Peyer's patches were sampled from cynomolgus monkeys, and the lymphoid follicle and germinal center areas and percentages of CD3- and CD20-positive areas were calculated. All the organs analyzed showed large interindividual variations in the sizes of lymphoid follicles and germinal centers. Lymphoid follicle in the spleen, submandibular lymph nodes and Peyer's patches showed no marked difference in size. Germinal center size in the mesenteric lymph nodes and Peyer's patches were significantly smaller than those in the spleen. Areas containing T cells were largest in the lymph nodes, while those containing B cells were largest in the spleen and Peyer's patches. The mean size of the splenic lymphoid follicle in cynomolgus monkeys is larger than that in rats and similar to that in humans. Based on the large individual variation and the characteristics of lymphoid organs, it is important to use cynomolgus monkeys in standard toxicity studies. Taking advantage of the characteristics of each species enables reliable evaluation of the immunologic system in standard toxicity studies.     

Perinatal development of lymphoid tissue and its associated epithelium in the ovine pharyngeal tonsil: A morphological study

The perinatal development of lymphoid tissue and its associated epithelium in the pharyngeal tonsil of sheep was investigated by light and electron microscopy. The lymphoid cells first appeared in the subepithelium in a scattered form at about 92 days gestation. These cells proliferated rapidly during the last trimester of gestation, and had formed many dense aggregates at the time of parturition. At birth, the epithelium overlying the aggregates was extensively infiltrated with lymphocytes and showed early transformation of follicle-associated epithelium. The lymphoid tissue and its associated epithelium did not, however., fully develop until after birth, when well-differentiated follicle-associated epithelium and lymphoid follicles with vigorous lymphopoiesis were present. In l–2 week old lambs, these structures were ultrastructurally similar to those seen in adult sheep although their size was smaller. The results of this study suggest that the lymphoid tissue of the ovine pharyngeal tonsil and its associated epithelium are morphologically ready to cope with antigens in the extra-uterine environment at birth, but that their full development and maturation are dependent on postnatal antigen stimulation.     

Perinatal development of lymphoid tissue and its associated epithelium in the ovine pharyngeal tonsil: a morphological study

The perinatal development of lymphoid tissue and its associated epithelium in the pharyngeal tonsil of sheep was investigated by light and electron microscopy. The lymphoid cells first appeared in the subepithelium in a scattered form at about 92 days gestation. These cells proliferated rapidly during the last trimester of gestation, and had formed many dense aggregates at the time of parturition. At birth, the epithelium overlying the aggregates was extensively infiltrated with lymphocytes and showed early transformation of follicle-associated epithelium. The lymphoid tissue and its associated epithelium did not, however, fully develop until after birth, when well-differentiated follicle-associated epithelium and lymphoid follicles with vigorous lymphopoiesis were present. In 1-2 week old lambs, these structures were ultrastructurally similar to those seen in adult sheep although their size was smaller. The results of this study suggest that the lymphoid tissue of the ovine pharyngeal tonsil and its associated epithelium are morphologically ready to cope with antigens in the extra-uterine environment at birth, but that their full development and maturation are dependent postnatal antigen stimulation.     

Secondary lymphoid areas in calf ileal Peyer's patch

The secondary lymphoid tissues appear in sheep ileum after involution of ileal Peyer's patch (PP). However, the existence of the secondary lymphoid tissues before involution of ileal PP has not yet been studied. We examined morphological characteristics of the full length of calf ileal PP using gross and microscopic anatomical techniques. Most areas of ileal PP consisted of densely packed lymphatic follicles contained very few follicular T-cell and associated with only scant interfollicular areas. However, the proximal end of ileal PP consisted of widely dispersed lymphatic follicles contained many follicular T-cell and associated with large interfollicular areas. The histological architectures of the proximal end of ileal PP strongly resembled those of the secondary lymphoid tissue in calf.     

Preparation, purification and characterization of bovine Peyer''s patch leukocytes.

An enzymatic technique for isolating bovine Peyer's patch leukocytes was developed. Enzymatic dissociation of Peyer's patches yielded a cell population rich in T and B lymphocytes containing 6-10% adherent accessory cells, as defined by morphology and nonspecific esterase staining. Analysis of Peyer's patch lymphocytes, using sheep erythrocyte rosetting and immunofluorescence with conventional antisera and monoclonal antibodies, showed that leukocytes were approximately 45% T cells and 25% Ig-positive B cells. The cell population contained functionally active T and B lymphocytes which proliferated in response to T and B cell mitogens and to exogenous human recombinant interleukin-2. The observed differences in patterns of Peyer's patch leukocyte responsiveness to these mitogens suggest some cellular heterogeneity of the bovine Peyer's patch.     

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.     

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.     

Lymphocyte migration studies

For maintenance of immunity and tolerance, the organs and tissues of the organism are connected by migrating lymphoid cells. Understanding lymphocyte migration is essential for many disorders and diseases-- especially in the mucosa-lined organs. Detailed analyses of migrating lymphocytes have been performed in many species, especially in laboratory animals. However, important experiments in lymphocyte migration have been carried out in large animals, for example sheep, cattle and pigs. These species allow experimental procedures like in situ-organ labelling, lymphocyte retransfusion studies or lymph vessel cannulations. Such studies have made an important contribution to the understanding of the overall principles of lymphocyte migration especially in the mucosal immune system. Major results on the specific migration of na?ve and memory T cells through lymphoid organs, the re-distribution of gamma/delta T cells in the intestinal immune system and the emigration of newly produced B cells from the ileal Peyer's patches have been obtained in large animals. Since there are growing numbers of markers for large animals, and molecular biology methods are available in these species, experiments in large animals will be an essential tool for the understanding of lymphocyte migration especially in mucosal organs.     

Comparative studies on the secondary lymphoid tissue areas in the chicken bursa of Fabricius and calf ileal Peyer's patch

The chicken bursa of Fabricius and calf ileal Peyer's patch are thought to be the primary lymphoid organs of B cell development. In the bursa, the existence of secondary lymphoid tissue, called the diffusely infiltrated area, has been recognized. Recently, we have found the presence of a region of secondary lymphoid tissue in the ileal Peyer's patch at the period of the most rapid growth of this organ. In this study, we compared the development of these secondary lymphoid tissue regions in the bursa and ileal Peyer's patch histologically. Before hatching, lymphatic follicle formation occurred in the bursa, but not in the diffusely infiltrated area, where only a small number of lymphoid cells were found. However, during fetal calf development, lymphatic follicle formation occurred not only in the primary lymphoid organ but also in the secondary lymphoid tissue regions. Therefore, the prenatal development of the secondary lymphoid tissue regions of the bursa and ileal Peyer's patch were distinct. After hatching, formation of the germinal center, which contained many CD4⁺ cells, was observed in the diffusely infiltrated area of the bursa. After birth, many CD4⁺ cells and IgG mRNA expression were observed in the lymphatic follicle of the secondary lymphoid tissue regions in the ileal Peyer's patch, but rarely in the ileal Peyer's patch lymphatic follicles. The change of character of these secondary lymphoid tissue regions at the postnatal stage might be dependent on external antigens.