The effect of hormonal bursectomy on the histological development of follicles in the embryonal bursa of Fabricius in the chicken

Forty chick embryos were hormonally bursectomized by dipping the eggs in testosterone propionate on day 3 of incubation. On day 14, 15, 16 and 17 of incubation the chick embryos were sacrificed and bursae of Fabricius were examined histologically. Besides depletion of bursal follicles and polypoid overgrowth of bursal epithelium, invagination of epithelial buds into the mesenchymal layer failed to occur and budding of bursal follicles into the bursal lumen was observed. This budding of bursal follicles could be caused by an alteration in the subepithelial mesenchymal layer caused by the hormonal treatment.     

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.     

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.     

Chemical bursectomy of chickens with colchicine applied to the anal lips.

To induce chemical bursectomy, 30 microliter colchicine dissolved in saline solution (1 mg/ml) was applied on the anal lips of White Leghorn chickens once daily for four consecutive days after hatching. Histologic characteristics of the bursa of Fabricius, spleen, thymus, cecal tonsils, and rectal wall were studied 1-7 days after hatching. Total necrosis of the lymphoid cells and the follicle-associated epithelium in the bursa was observed during the four days of colchicine application. The bursal stroma remained unchanged, and only minor changes were found in the interfollicular surface epithelium. After colchicine application ceased, some regeneration of the epithelium, as evidenced by small epithelial buds, was found. At the end of the observation period the epithelial buds were often covered by the follicle-associated epithelium, which was capable of phagocytizing carbon. However, practically no lymphoid repopulation was seen in the buds. Since this method of colchicine application had no direct effect on other lymphoid organs or on the survival or weight of the chickens, this bursectomy model seems to be a new tool for use in studies of bursal function.     

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.     

Clones of B lymphocytes in individual follicles of the bursa of Fabricius

To discover whether individual bursal follicles can contain clones of B lymphocytes, we estimated the numbers of lymphoid cell precursors populating single follicles in two types of chicken chimera. The first type was produced by establishing parabiotic connections between blood vessels of embryo chorioallantoic membranes. Under these conditions, and most likely during normal development, most follicles are populated by more than one, but less than ten, precursor cells. However, in a second type of chimera, a cyclophosphamide-treated chick reconstituted with normal bursal cells, most follicles in the reconstituted bursa are clonal (their lymphocytes are derived from a single precursor cell). Individual follicles can readily be isolated from bursae of reconstituted birds and should be useful in studies of B cell development.     

Alkaline Phosphatase in the Developing Bursa of Fabricius

The ontogeny of alkaline phosphatase in the bursa of Fabricius was studied by histochemical and biochemical methods. According to the quantitative determinations, the activity of alkaline phosphatase increased from the 11th to 17th day of incubation--that is, during the time of the lymphoid follicle formation in the developing bursa. The activity was localized in the mesenchymal tissue surrounding the lymphoid follicles. Testosterone given in ovo prevented the appearance of alkaline phosphatase in the bursal mesenchyme but had no effect on the activity of the embryonic liver. In contrast, in ovo treatment with cyclophosphamide had no effect on the alkaline phosphatase in the bursa. By using transplantation of embryonic bursal stem cells, it was further shown that, in contrast to cyclophosphamide, testosterone destroys the capacity of the bursa to serve as a differentiation site for the B-cell lineage. The results indicate that testosterone affects the stromal cells of the bursa, whereas cyclophosphamide destroys only the lymphoid population undergoing differentiation and leaves the bursal stroma intact.     

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.     

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.     

Comparative anatomy of mammalian conjunctival lymphoid tissue: a putative mucosal immune site

Organized mucosa-associated lymphoid tissue (O-MALT) is defined by mucosal lymphoid follicles with unique overlying lymphoepithelia, and classically appears in tissues with a simple columnar epithelium. Within follicle-associated epithelium, goblet cells are characteristically absent, replaced by ultrastructurally distinct antigen-absorptive cells, termed M cells (or microfold cells) for the appearance of their apical cell membranes. To determine if mammalian conjunctiva, with its stratified squamous epithelium, can be considered as a site of O-MALT, we compared the light and electron microscopic anatomy of conjunctiva from fourteen species of non-human adult mammals, and the conjunctiva of human adults harvested at autopsy. Lymphoid follicles in the conjunctiva were demonstrated in all mammals studied except for mice and rats. In those mammals with conjunctival lymphoid follicles, the follicle-associated conjunctival epithelium was notable for an absence of goblet cells. Transmission electron microscopy demonstrated an intimate association of lymphocytes with surface epithelial cells, but epithelial cell morphology was uniform overlying the follicle, and other ultrastructural features of M cells were absent. Therefore, conjunctival lymphoid follicle-associated stratified squamous epithelium demonstrates some but not all features of O-MALT lymphoepithelia. Further studies are necessary to determine what role conjunctival lymphoid tissue may play in mucosal immunity.     

Avian B cell precursors: surface immunoglobulin expression is an early, possibly bursa-independent event

The avian bursa of Fabricius contains about 1 X 10(4) discrete follicles, each of which is colonized by a small number of lymphoid progenitor cells during embryonic life. We have previously shown (J.R.L. Pink et al., Eur. J. Immunol. 1985. 15:617) that all, or almost all B cell progenitors in the bursae of 4-day-old chicks express cell surface IgM. In this report, we have analyzed the distribution of cell surface (s)IgM-1 allotypes within individual follicles of (M-1a/M-1b) allotype heterozygous birds. Although the majority of follicles contained a mixture of sIgM-1a+ and sIgM-1b+ cells, a significant proportion of isolated follicles contained exclusively sIgM-1a+ or sIgM-1b+ cells. Statistical analysis of the frequency of such "M-1a" and "M-1b" follicles demonstrated that the sIg+ B cells in the bursae of 4-8-week-old birds are derived from 2-4 allotypically committed precursor cells per follicle. Since we have previously shown that each bursal follicle is colonized by 2-5 pre-bursal stem cells, these cells must be committed to the eventual expression of one or other allotypic haplotype before they have undergone extensive proliferation within the bursa. In addition, we show that almost all B progenitor cells from the bursae of chicks which had been allotype suppressed as embryos were committed to synthesis of the nonsuppressed allotype, showing that this commitment was essentially complete at the time of suppression (i.e. before 19 days of incubation). Finally the bone marrow of 16-day embryos was used to reconstitute the bursal lymphocytes of cyclophosphamide-treated host embryos. Reconstitution was inhibited by anti-Ig antiserum indicating that most 16-day embryonic BM-derived bursal cell precursors also express sIgM. These results raise the possibility that expression of sIgM may be controlled by a "biological clock" rather than by any inductive capacity of the bursal microenvironment. Furthermore, these results provide further evidence that in normal birds a self-renewing sIg+ B cell population in the hatched chicken is the sole source of B cells in the adult.     

Lymphocyte requirement for the functional development of follicle-associated epithelium

Follicular development in the bursa of Fabricius was disrupted by testosterone treatment and chorioallantoic membrane grafting. Analysis by a quantitative histological technique demonstrated that testosterone treatment causes a dose dependent delay in development by inhibiting lymphoid proliferation. Inhibition of lymphoid development prevented the development of carbon transport ability by follicle associated epithelium. Reconstitution of follicular development by grafting treated bursae to the chorioallantoic membrane of untreated hosts results in the development of functional follicle associated epithelium. These results establish the lymphoid requirement for the development of transport ability by the follicle-associated epithelium.     

Immunoglobulin-bearing stem cells for clones of B (bursa-derived) lymphocytes

Treatment of neonatal chickens with cyclophosphamide depletes bursal lymphocytes while maintaining the bursal epithelium intact. The bursae of normal young chickens contain "bursal stem cells" which can reconstitute the lymphoid compartment in the bursa of the cyclophosphamide-treated recipient. Using bursal stem cells from IgM allotype-heterozygous donors we show that most bursal follicles in the reconstituted host are colonized by single stem cells which are committed to the expression of one or other IgM allotype. In addition we show that the reconstituting bursal stem cells express allelically excluded surface IgM at the time of transfer. Our results suggest that B lymphocyte numbers in hatched chickens are maintained by self-renewal of committed precursors rather than by de novo production from multipotential stem cells.     

Lymphocyte depletion in ileal Peyer's patch follicles in lambs infected with Eimeria ovinoidalis.

A total of 14 lambs were experimentally infected with Eimeria ovinoidalis in two separate experiments in two consecutive years. Nine lambs served as uninoculated controls. Material was collected from the ileum 2 weeks after infection in eight lambs and 3 weeks after infection in six lambs. Lambs examined 2 weeks after infection had normal follicles. After three weeks, the follicle-associated epithelium covering the lymphoid follicles of the ileal Peyer's patches showed fusions with adjacent absorptive epithelium, focal hyperplasia, and occasionally necrosis. Macrogametes, microgamonts, and oocysts were often found in the follicle-associated epithelium and the dome region. Various degrees of lymphocyte depletion were present in the ileal lymphoid follicles in all six infected lambs 3 weeks after infection, and four lambs had decreased follicle size. Reduced staining for leukocyte common antigen (CD45), B-cell markers, and the proliferation marker Ki-67 was present in these lambs. Application of the terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling method for apoptotic cells revealed decreased staining in the ileal lymphoid follicles 3 weeks after infection. A marker of follicular dendritic cells, 5'- nucleotidase, showed increased reactivity, probably due to condensation of reticular cells following loss of follicle lymphocytes. Reduced staining for carbonic anhydrase in the follicle-associated epithelium and the domes was present in all six lambs examined 3 weeks after infection, indicating decreased production of carbonic anhydrase-reactive 50-nm particles and a decreased lymphoproliferative stimulus. In conclusion, the present study shows that severe E. ovinoidalis infection in lambs causes lesions of the follicle-associated epithelium and may result in lymphocyte depletion and atrophy of the ileal Peyer's patch follicles.     

Structure of lamina propria lymphoid follicles and associated epithelium in the gastric mucosa during Helicobacter pylori infection in ulcer-bearing Mongolian gerbils

To develop a gerbil model of Helicobacter pylori-induced chronic active gastritis comparable in severity to human lesions, we made acetic acid-induced ulcer in the anterior antral wall and concurrently challenged 1 x 10(8) colony-forming units bacteria per os. At 30 and 60 days after inoculation, the number of viable bacteria colonizing on the surface epithelium of the gastric mucosa was larger in ulcer-bearing animals compared to non-bearing ones. Furthermore, in the former animals, neutrophil and mononuclear cell infiltration as well as lymphoid follicle formation in the lamina propria was more prominent. Electron microscopically, lymphoid follicle-associated epithelium displayed specialized structures. Namely, brush cells interposed between mucous epithelial cells and characterized by prominent microfilament bundles and many apical vesicles or caveola specifically embraced the cluster of intraepithelially invading lymphocytes and macrophage-like cells by the attenuated cytoplasm in an analogous manner to M cells in Peyer's patches. The present study has demonstrated that ulcer formation enhances both H. pylori colonization and lamina propria lymphoid follicle formation and suggested that follicle-associated epithelium might play roles in the delivery of intraluminal antigen.     

Lymphocyte Depletion in Ileal Peyer’s Patch Follicles in Lambs Infected with Eimeria ovinoidalis

A total of 14 lambs were experimentally infected with Eimeria ovinoidalis in two separate experiments in two consecutive years. Nine lambs served as uninoculated controls. Material was collected from the ileum 2 weeks after infection in eight lambs and 3 weeks after infection in six lambs. Lambs examined 2 weeks after infection had normal follicles. After three weeks, the follicle-associated epithelium covering the lymphoid follicles of the ileal Peyer’s patches showed fusions with adjacent absorptive epithelium, focal hyperplasia, and occasionally necrosis. Macrogametes, microgamonts, and oocysts were often found in the follicle-associated epithelium and the dome region. Various degrees of lymphocyte depletion were present in the ileal lymphoid follicles in all six infected lambs 3 weeks after infection, and four lambs had decreased follicle size. Reduced staining for leukocyte common antigen (CD45), B-cell markers, and the proliferation marker Ki-67 was present in these lambs. Application of the terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling method for apoptotic cells revealed decreased staining in the ileal lymphoid follicles 3 weeks after infection. A marker of follicular dendritic cells, 5′- nucleotidase, showed increased reactivity, probably due to condensation of reticular cells following loss of follicle lymphocytes. Reduced staining for carbonic anhydrase in the follicle-associated epithelium and the domes was present in all six lambs examined 3 weeks after infection, indicating decreased production of carbonic anhydrase-reactive 50-nm particles and a decreased lymphoproliferative stimulus. In conclusion, the present study shows that severe E. ovinoidalis infection in lambs causes lesions of the follicle-associated epithelium and may result in lymphocyte depletion and atrophy of the ileal Peyer’s patch follicles.     

M-cells are damaged and increased in number in inflamed human ileal mucosa

Ileocolonoscopy and biopsies of patients with spondylarthropathy reveal gut inflammation in 62% of cases. In order to better understand the pathogenetic mechanisms of spondylarthropathy-related gut inflammation, the follicle-associated epithelium was examined. Biopsies from nine controls and 18 patients with spondylarthropathy were studied by electronmicroscopy. Membranous (M) cells were investigated in normal and inflamed ileum. In normal mucosa, M-cells were scarce whereas in inflamed mucosa their number was increased (up to 24% of follicle-associated epithelial cells). They showed a thin rim of cytoplasm covering groups of lymphocytes. In chronic ileitis, necrotic M-cells, rupture of M-cells and lymphocytes entering the gut lumen were observed. The bursting of M-cells at the top of the lymphoid follicles leads to interruption of the gut epithelial lining and gives the luminal content access to the lymphoid tissue. This pathogenetic mechanism may cause aphthoid ulcers.     

Comparison of the in situ changes in lymphoid cells during infection with infectious bursal disease virus in chickens of different ages.

In situ immunocytochemical staining was used to characterise leukocyte changes and determine tropism of infectious bursal disease virus following infection of neonate and 3-week-old chickens. In the bursae of both groups, massive replication of the virus, a rapid depletion of B cells and an influx of CD4(+) TCR-alphabeta(1)(+) and CD8 (+) TCR-alphabeta(1)(+) cells was detected within 4 days post-inoculation. Leukocyte changes in the spleen, thymus and Harderian gland were similar in both groups. From 8 days post-inoculation onwards all the lymphoid organs became repopulated with leukocytes and tissue architecture began to be slowly restored. Virus neutralizing antibodies developed more slowly in neonate birds and at 21 days post-inoculation the titres were much lower compared to older birds. Lack of clinical signs in neonate chickens was neither due to a failure to respond to the virus, to recruit leukocytes to the infected tissues nor to a lack of viral replication.     

Locally applied testosterone is a novel method to influence the development of the avian bursa of Fabricius

Bursa of Fabricius is a primary lymphoid organ in the birds and its microenvironment is responsible for the B cell maturation. The epithelial anlage develops through epithelial-mesenchymal interaction into which -as a third party - hemopoietic cells immigrate. Chemical bursectomy can be made by dipping or injecting eggs by testosterone solution. Both methods of treatments compromise the development of the whole embryo. Heparin beads of 70-150 µm in diameter were soaked in 5% testosterone solution and implanted into the tail bud of 4 day old chicken embryos before the bursal anlage began to develop at embryonic day 5. The locally applied testosterone inhibited the differentiation of the bursal secretory dendritic cell (BSDC) precursors, which were the inducer of the follicular epithelial bud formation. Testosterone treatment did not blocked the entering of B cells and other hemopoietic cells into the bursal mesenchyme, but in the absence of BSDC precursor cells the dendroepithelial microenvironment is not established, subsequently B cell precursors are lodged only in the bursal mesenchyme. This method is suitable for locally introducing other regulatory molecules, involved in bursal development.     

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.