Morphological and functional differentiation of the surface epithelium of the bursa Fabricii in chicken

Morphological and functional differentiation of the mucosal surface epithelium of the bursa Fabricii was studied in White Leghorn chicken fetuses and newly hatched chickens. First signs of differentiation towards two types of epithelial cells appeared on the thirteenth day of incubation: The apical cells of the epithelial buds projected towards the lumen, and an increase in the number of Golgi regions was observed in the epithelial cells between the buds. On day 15 the follicle-associated epithelium contained small apically situated vacuoles, and large mucin granules appeared in the interfollicular surface epithelium. Towards the day of hatching both epithelial cell types were arranged to a monolayered or pseudostratified cylindrical epithelium. The follicle-associated epithelium had invaginations and small vacuoles in the apical cytoplasm, whereas the interfollicular surface epithelium had numerous microvilli on its apical surface and large mucin granules in the apical cytoplasm. In functional studies, endocytosis of colloidal carbon was demonstrated in four out of ten 19-day-old fetuses and in all chickens studied immediately after hatching.     

Histochemical characterization of chicken lymphoid tissues

Thirteen enzyme activities were studied histochemically in the chicken bursa of Fabricius, thymus and spleen. The bursal epithelium contained a high activity of succinate, lactate and glucose-6-phosphate dehydrogenases and of ATPase. Activities of these enzymes were markedly lower in the follicle-associated epithelium than in the other parts of the bursal epithelium. A high activity of α-esterase occurred in the follicle-associated part in contrast to the other parts of the epithelium. The histochemical differentiation of the follicle-associated epithelium started at the time of hatching and was completed within a week. ATPase positive cells were observed in the interfollicular space of the bursa a few days before and after the hatching. The nature of these cells remained unclear. In the thymus, a good activity of lactate and glucose-6-phosphate dehydrogenases was observed in the area between the cortex and medulla, possibly indicating presence of endocrine cells. In the spleen no specific location of enzyme activities was observed.     

Pinocytosis by epithelium associated with lymphoid follicles in the bursa of fabricius,appendix, and Peyer's patches. An electron microscopic study

The fine structure and micropinocytotic capabilities of epithelial cells closely associated with lymphoid follicles in the chicken bursa of Fabricius, rabbit appendix, and mouse Peyer's patch were compared. Epithelial cells capable of transporting ferritin and India ink tracers from the lumen were demonstrated in all three locations. Epithelial cells not associated with lymphoid follicles in the bursa and appendix did not express pinocytotic activity. Lymphoid cells were identified in bursal epithelium of chick embryos as early as the twelfth day of incubation. These lymphoid cells were smaller than typical bursal lymphocytes, had dense cytoplasm, numerous cytoplasmic projections, and prominent nucleoli. The small lymphoid cells were first demonstrable at a time in incubation during which lymphoid stem cells have been shown to migrate into the bursal epithelium. Lymphoid cells were seen earlier than the specialized follicle-associated epithelium. It is concluded that specialized pinocytotic follicle-associated epithelium does not induce initial migration of stem cells into areas along the intestinal tract, but that this transepithelial pinocytotic flow of intestinal contents after birth may provide a significant stimulus for attraction, proliferation and egression of lymphocytes.     

Cyclophosphamide- and testosterone-induced alteration in chicken bursal stroma identified by monoclonal antibodies.

Both cyclophosphamide (Cy) and testosterone propionate (TP) treatments ablate B cells in chickens. Essential bursal microenvironmental elements, however, are altered or lost following TP treatment, while bursae from Cy-injected birds can be reconstituted with donor precursors. These two models can thus be utilized to distinguish which bursal stromal molecules are functionally most important in the specific microenvironment of this organ. Monoclonal antibodies (mAb) reactive with non-lymphoid components of the chicken bursa of Fabricius have been used to examine bursal sections from birds treated with Cy or TP. Molecules have been identified on the epithelial buds and follicle-associated epithelium (FAE) that are enhanced following Cy treatment (MUI-52 and 58) and are absent in TP-treated birds. The expression of these molecules may correspond to the ability of Cy-treated but not TP-treated bursae to attract lymphoid precursors. Molecules have also been identified on cells in the subepithelial mesenchymal layer (MUI-63, 65 and 75). These cells interact with the surface epithelium (sEp) prior to epithelial bud formation, an interaction which appears to be TP sensitive. Additionally, two potentially important molecules have been identified in the bursal medulla (MUI-54 and 72) which may have an interactive role with developing B lymphocytes.     

The behavior of bursal lymphoid follicle-associated cells after treatment with testosterone

Administration of androgens produces damage in lymphoid tissue and in the bursa of Fabricius. After IM administration of 5 mg of testosterone propionate (TP) beginning at hatching and continued during the following 4 days, a significant reduction in the bursal weights is observed. Histologically, an increase in the connective tissue is observed and cystic formations are also found. In all cysts examined, there is continuity of the cystic lumen with the free surface. The follicle-associated epithelial (FAE) cells are on the bottom of the pseudocysts and form a separation between the pseudocystic cavity and the lymphoid tissue which is still further inwards. These cells do not lose their esterase activity, even though they are often flattened. Furthermore, they disappear in the pseudocysts deprived of lymphoid tissue. A new hypothesis is advanced that the FAE cells originate from the mesenchyme with differentiation in the histocytic line.     

On the role of the lymphoid follicle-associated areas in the organization of the bursal follicle in the cloacal bursa in birds

The present paper contains data on the morphological modifications present in chicks which, after treatment with testosterone lasting 10 days (from the seventh to the seventeenth days after hatching), were killed on the 30th, 35th, 40th, 50th and 70th day of life; this interval of time might theoretically have allowed a gradual re-establishment of the bursal architecture. Examination of the bursae of Fabricus of the animals treated showed greatly varying pictures. The 2 main types of behaviour were the following: the histological pattern of certain bursae had been completely restored to normal. Others did not act in the same way but wide areas of their plicae proved to be devoid of lymphoid tissue and well-formed lymphoid follicles were observed only in certain places. These 2 kinds of behaviour, and particularly what we observed in the second case, might suggest that, since the lymphoid follicle-associated epithelial cells are the last to disappear after treatment with testosterone propionate and the follicles are restored only where these cells remain, the lymphoid follicle-associated epithelial cells may be considered to be responsible for the re-establishment of the bursal follicle.     

Development of the follicle-associated epithelium and the secretory dendritic cell in the bursa of fabricius of the guinea fowl (Numida meleagris) studied by novel monoclonal antibodies

Two stromal elements, follicle-associated epithelium and secretory dendritic cells of the bursa of Fabricius were studied by light microscopy and two novel MAbs, that were produced against splenic cell suspensions of guinea fowls. Both antigens recognized by these MAbs, designated GIIF3 and NIC2, are localized in the cytoplasm of the stromal cells, and their molecular weights are 50 and 30 kD, respectively. During embryogenesis the GIIF3 and NIC2 cells emerge in the mesenchyme of the folds before follicle formation. The GIIF3 and the NIC2-positive cells accumulate under the surface epithelium of the plicae and migrate into the epithelium, that precedes the bud-formation. From the bud, the GIIF3-positive cells migrate up to the luminal surface, and they transform to distinct, highly polarized follicle-associated epithelial cells. Single GIIF3-positive cells are also present in the interfollicular epithelium. The NIC2 MAb recognized mesenchymal cells harbor in the lymphoepithelial compartment of the folliculus, and they elaborate cytoplasmic granules. Around Day 20 of embryogenesis large amount of NIC2-positive substance appear extracellularly in the medulla and around it. This period well correlates with the starting up of the bursal functions; clonal expansion of B cells, and generation of immune repertoire. After hatching the NIC2 stainability diminishes, and it is restricted to the medullary bursal secretory dendritic cells. The NIC2-positive, possibly elderly bursal secretory dendritic cells, are capable for migration into the follicle-associated epithelium. In eight-day old birds some cells of the follicle-associated epithelium reveals temporary NIC2 positivity, that may prove the transport of the follicle-associated epithelial cells into luminal direction. By 12 weeks of age the presence of NIC2-positive substance in the intercellular space of the FAE, rather than in the cells of FAE may indicate the termination of the transport of secretory substance. In conclusion, two types of mesenchymal cells enter the surface epithelium of the bursal folds. The GIIF3-positive cells appear on the luminal surface of the follicles and occupy the place of the follicle-associated epithelial cells. The NIC2-positive cells become secretory in nature and differentiate to bursal secretory dendritic cells. The follicle formation possibly, requires the joint presence of both GIIF3 and NIC2 cells in the epithelium.     

Bursal and postbursal stem cells in chicken. Functional characteristics

Cyclophosphamide (CY)-treated or surgically bursectomized, CY-treated 3-day old chicks were injected with bursa or bone marrow cells from donors of different ages. Cell recipients and donors were isogeneic at the major histocompatibility locus. Antibody responses to sheep red blood cells and Brucella abortus, and microscopic morphology of spleen and bursa were assessed 5–6 weeks after the cell transplantation. Relative weight of the bursa was found to be a reliable indicator of the restoration of bursal structure. The results indicate that stem cells or progenitor cells for the B cell line in chicken can be divided into a bursal stem cell and a postbursal stem cell. Both of these cell types are effective in restoring antibody formation of CY-treated chickens. Bursal stem cells restore the bursal morphology; they are not capable of further maturation without the influence of the bursal microenvironment. This influence is not effected by bursa in a cell impermeable diffusion chamber; actual contact with the bursal stroma is necessary. Bursal stem cells are also capable of restoring the formation of germinal centers in the spleen of CY-treated chickens. Postbursal stem cells do not restore bursal structure and they do not need the bursal micro-environment for further maturation. They also have no clear effect on the formation of germinal centers in the spleen. Bursal stem cells are found in the bursa during the first few weeks after hatching. Postbursal stem cells start to appear in the involuting bursa and at the same time, in the bone marrow also. They are found as the majority of cells in the bursa and bone marrow after at least the 10th week following hatching. Early postbursal stem cells have already passed the education given by the bursa, but have not yet totally lost their capacity to induce germinal centers in the spleen. They restore germinal center formation even in surgically bursectomized recipients, demonstrating that presence of bursal follicles is not necessary for the production of germinal centers. The findings are discussed to stress the significance of two equally important factors in the development of immunity: the lymphoid cells themselves and, at each stage ontogeny, the proper microenvironment for their further function and maturation.     

The influence of embryonic testosterone treatment on bursal epithelial pinocytotic activity

Developing White Leghorn chicks were treated with testosterone propionate $\text{in}$$\text{ovo}$ to effect a partial chemical bursectomy. After hatching, birds were administered carbon particles or horseradish peroxidase via the cloaca, and the transport of these tracers by the bursal epithelium was examined. Uptake of both tracers was inhibited in the treated when compared to untreated birds. The degree of development of interaction between bursal epithelium and underlying lymphoid tissue appeared to play a major role in determining ability of the epithelium to transport these tracers. Epithelium associated with follicles possessing medullary and cortical divisions was able to transport, while intraepithelial follicles or regions of the epithelium overlying diffuse lymphoid tissue did not transport either carbon or horseradish peroxidase. Bursal function in regional defense of the gut may depend on a local interaction of bursal epithelium with lymphoid, and perhaps other cell types.     

Involution of the chicken bursa of Fabricius: a light microscopic study with special reference to transport of colloidal carbon in the involuting bursa

The involution of the bursa of Fabricius in White Leghorn chickens and the transport of colloidal carbon in the involuting bursa were studied on light microscopy. In chickens, from newly hatched to 23.5 weeks of age, the bursa was weighed, its histology was described, and the mitoses in the cortical and medullary compartments of the lymphoid follicles were counted. Decrease in bursal weight and in the number of mitoses were observed between 10 and 16 weeks of age. During week 17.5, the follicle-associated epithelium began to lose its endocytic capability, and mucin droplets appeared in the follicular medulla initiating the large mucoid cysts that were seen in the later phases of involution. The involutionary process was almost completed by week 23.5, when the bursa appeared as a fibrotic residue without intact lymphoepithelial structures. The particulate tracer used, colloidal carbon, was not observed to be transported to other organs from the bursa. The present study gives further support to the idea that after completing its central immune function in 6 weeks [21] the bursa, as estimated histologically, still possesses capacity to function like a peripheral lymphoid organ until 16 weeks of age.     

Abnormalities associated with the bursal microenvironment in spontaneous dysgammaglobulinemia of UCD line 140 chickens

UCD line 140 chickens have been previously reported to develop a syndrome of spontaneous 7s immunoglobulin deficiency and the presence of autoantibodies. Earlier studies demonstrated that these inbred birds have normal peripheral blood T and B cell numbers; they also respond normally to allogeneic stimulation. Although the 7s immunodeficiency does not manifest itself until several months of age, line 140 birds have a premature degeneration of bursa. Because of the recent development of monoclonal reagents specific for bursal elements, including surface epithelium, basement membrane associated epithelium, follicle associated epithelium, and lymphoid subpopulations, we have examined line 140 and control birds for the expression of bursal epithelial cell antigens. Line 140 birds, in contrast to control chickens, have a dramatic early alteration in the expression of an epithelial cell marker in the bursa, thymus, and intestine. Moreover, to further address this issue, we transplanted bursa from 10-day embryos onto the chorioallantoic membrane, a privileged site. Bursae from control birds became abnormal when transplanted onto line 140 CAM; they remained normal when transplanted among several control chicken lines. In contrast, line 140 bursa remained abnormal independent of the transplant procedure. Due to the marked bursal abnormality observed specific to the dysgammaglobulinemia chicken line, we propose that the microenvironmental features of line 140 bursa may predispose these birds to the development of humoral immunodeficiency and autoantibodies.     

The development of unusual B-cell functions in the testosterone-propionate-treated chicken.

Chickens were treated with 4 mg of testosterone propionate on the twelfth day of embryonic life. Bursal remnants of testosterone-treated chickens were very small in size and had very few or no bursal follicles: the lymphoid tissue was replaced substantially by fibrosis. Testosterone-treated chickens formed almost exclusively IgM antibodies to sheep red blood cells and influenza virus, whereas no IgM or IgG response to Brucella abortus or Salmonella pullorum, and no IgG response to sheep red blood cells was demonstrable. Surgical removal of bursal remnants of testosterone-treated chickens at hatching did not significantly affect IgM response to sheep red blood cells. These B-cell functions of testosterone-treated chickens were not improved by addition of T cells, as shown by adoptive cell transfer experiments. Thus, there appears to be an unusual type B-cell development which is independent of the bursa of Fabricius.     

The bursal microenvironment: phenotypic characterization of the epithelial component of the bursa of Fabricius with the use of monoclonal antibodies.

Monoclonal antibodies were raised against newborn chick bursa of Fabricius, and here we describe two antibodies, BEP-1 and BEP-2, which react selectively with the epithelial component of the bursa of Fabricius. In previous studies, using quail chick chimeric bursas, we have demonstrated that the epithelium of the bursal rudiment, presumably of endodermal origin, gives rise to the epithelium lining the bursal lumen, the basement membrane-associated epithelium and the network of reticular cells of the medulla, while the interfollicular connective cells are derived from the mesoderm. When tested in indirect immunofluorescence assay on bursa tissue sections or cell suspensions, BEP-1 reacts with a surface antigen present on all the epithelial cells of the bursa and could be used as a marker for this cell lineage. BEP-2 binds to an intracytoplasmic antigen that is present in about 5% of cells, representing the epithelial cells, and which is excreted in the medulla. BEP-2 also reacts with the epithelial cells of the thymic medulla and with the mucin-secreting goblet cells of the intestinal villi. A rabbit antiserum raised against human cytokeratin gives a different pattern of reactivity on bursal tissue compared to BEP-1 and BEP-2, tentatively suggesting that these two antibodies do not bind to keratin-like molecules. During ontogeny, BEP-1 reactivity appears in bursal epithelium from the early stages of bursal ontogeny (8 days). BEP-2 reactivity is detected around hatching time. BEP-1 and BEP-2 do not show any antigenic heterogeneity among the epithelial cells of the bursa.     

Scanning electron microscopy of the bursa of fabricius from normal and testosterone-treated embryos

Scanning electron microscopy (SEM) has revealed the presence of projecting follicles (PF) and button-like follicles (BLF) in the bursa of Fabricius. This study was designed to examine the embryonic bursa with SEM to ascertain which type of follicle appears first and to compare the SEM of the bursa from normal embryos with those having received testosterone propionate (TP) on the 11th day of incubation. The bursa of the latter embryos exhibits an arrested lymphoid development. PF appeared in normal embryos by 16 days and were well developed by 18 days of embryonic development. Inter-follicular epithelium was apparent by 21 days of embryonic development in normal embryos. On the other hand, bursal follicles and interfollicular epithelium failed to form in TP birds. The TP-birds exhibited a characteristic pebble-like epithelium which may attest to the regressive influence of TP on bursal epithelium or to an arrested stage of epithelial development. The PF may lead to the development of BLF or the BLF may be derived independently of PF.     

Dome epithelium and follicle-associated basal lamina pores in the avian bursa of fabricius

Background: The immunological role played by the avian bursa of Fabricius has been well established. Although numerous studies have also reported on the development and general morphology of this organ, some structure-function relationships still hae not been fully explained. Methods. Bursae from chickens at three developmental stages were removed and examined by scanning electron micropscopy. Routine preparation was used as well as sonication (Microdissection). Micrographs were used for qualitative morphological study and for quantitative morphometric analyses. Results: Routine SEM observations were similar to those previously reported in the literature. Sonicated specimens allowed topographical study of various levels of surface erosion. Two types of surface cells were observed: typical absorptive epithelium and follicle-associated epithelial (FAE) cells. Erosion of the dome surface epithelium revealed basal lamina pores n the region over the subepithelial lymphoid follicles. These pores were present at hatching. Morphometric analysis of dome and pore areas. Conclusions: Basal lamina pores may provide a communication route between the lympghoid follicles and the external environment via the FAE cells. Also, the close association between the FAE cells of the epithelial domes, the epithelial pores, the capillary complex of the previously described bursal-blood barrier, and the subepithelial lymphoid follicles could represent a morphological “pure complex” that matures early in posthatching development and may be related to the immunological function of the bursa. © 1995 Wiley-Liss, Inc.     

Bursa Fabricii as a peripheral lymphoid organ: Transport of various materials from the anal lips to the bursal lymphoid follicles with reference to its immunological

In tracer studies, various particulate and molecular materials applied on the anal lips of chickens were transported to the bursal lumen. From here some of the tracers were further transported into the bursal lymphoid follicles and colloidal carbon even to the bursal stroma. In immunization experiments, Brucella abortus organisms and SRBC were applied on the anal lips of 1-day-old, 4-week-old and 10-week-old chicks on 5 consecutive days. Although Brucella bacteria in tracer studies were not found in the bursal tissue, agglutinin response to Brucella was observed in the 4-week and 10-week groups as early as 3 days after the last application of this antigen. The response to SRBC was strong only in the oldest group of chickens. When stimulated continuously, the agglutinin titres reached their highest values after the 2nd week and then began to decrease to rather low levels. In the youngest age group, the anti-SRBC titres, however, remained low during the whole experimental period, and even in the 4-week group the anti-SRBC response was weak. Bursectomy, carried out at the age of 10 weeks, inhibited the agglutinin response to Brucella but affected only little the response to SRBC. It is concluded that by taking up antigens per anum the chicken possibly gains part of its basic immunity. In this immune response the bursa seems to play an important role at least with some antigens, and thus the bursa may have an immunofunction comparable with that found in peripheral lymphoid organs.     

Bursal fabricii as a peripheral lymphoid organ. Transport of various materials from the anal lips to the bursal lymphoid follicles with reference to its immunological importance

In tracer studies, various particulate and molecular materials applied on the anal lips of chickens were transported to the bursal lumen. From here some of the tracers were further transported into the bursal lymphoid follicles and colloidal carbon even to the bursal stroma. In immunization experiments, Brucella abortus organisms and SRBC were applied on the anal lips of 1-day-old, 4-week-old and 10-week-old chicks on 5 consecutive days. Although Brucella bacteria in tracer studies were not found in the bursal tissue, agglutinin response to Brucella was observed in the 4-week and 10-week groups as early as 3 days after the last application of this antigen. The response to SRBC was strong only in the oldest group of chickens. When stimulated continuously, the agglutinin titres reached their highest values after the 2nd week and then began to decrease to rather low levels. In the youngest age group, the anti-SRBC titres, however, remained low during the whole experimental period, and even in the 4-week group the anti-SRBC response was weak. Bursectomy, carried out at the age of 10 weeks, inhibited the agglutinin response to Brucella but affected only little the response to SRBC. It is concluded that by taking up antigens per anum the chicken possibly gains part of its basic immunity. In this immune response the bursa seems to play an important role at least with some antigens, and thus the bursa may have an immunofunction comparable with that found in peripheral lymphoid organs.     

Humoral immunodeficiency in chickens induced by chemical bursectomy with colchicine applied on the anal lips

Colchicine was applied on the anal lips of chickens on 4 consecutive days after hatching. At the ages of 4 and 10 weeks the agglutinin responses to Brucella abortus and to sheep red blood cells (SRBC) after intraperitoneal and per anum immunization were decreased in these animals. IgG and IgM antibodies to Brucella, measured by enzyme-linked immunosorbent assay (ELISA), were also diminished in 12-week-old chickens. The total concentration of plasma IgG, measured by the ELISA-inhibition method, was diminished whereas the IgM level was not influenced by the colchicine treatment. These results broadly resemble the findings after surgical bursectomy or cyclophosphamide treatment. It can be concluded that colchicine applied on the anal lips of chickens causes a long-lasting deficiency of humoral immunity and thus seems to be a useful new tool in the exploration of the humoral-immune functions of the chickens.     

Immune growth hormone (GH): localization of GH and GH mRNA in the bursa of Fabricius

Expression of growth hormone (GH) and GH receptor (GHR) genes in the bursa of Fabricius of chickens suggests that it is an autocrine/paracrine site of GH production and action. The cellular localization of GH and GH mRNA within the bursa was the focus of this study. GH mRNA was expressed mainly in the cortex, comprised of lymphocyte progenitor cells, but was lacking in the medulla where lymphocytes mature. In contrast, more GH immunoreactivity (GH-IR) was present in the medulla than in the cortex. In non-stromal tissues, GH-IR and GH mRNA were primarily in lymphocytes, and also in macrophage-like cells and secretory dendritic cells. In stromal tissues, GH mRNA, GH and GHR were expressed in cells near the connective tissue (CT) between follicles and below the outer serosa. In contrast, GH (but not GH mRNA or GHR), was present in cells of the interfollicular epithelium (IFE), the follicle-associated epithelium (FAE) and the interstitial corticoepithelium. This mismatch may reflect dynamic temporal changes in GH translation. Co-expression of GHR-IR, GH-IR, GH mRNA and IgG was found in immature lymphoid cells near the cortex and in IgG-IR CT cells, suggesting an autocrine/paracrine role for bursal GH in B-cell differentiation.