Nándor Nagy

Structure of the Avian Lymphoid System

Our ability to understand the function, pathology and regeneration ability of an organ system is handicapped without knowledge of its normal structure. Electron microscopy and, later, immunocytochemistry made it possible to extend our knowledge of the structure of cells and the communication between them. These novel techniques helped to recognize the avian dendritic cells. Contemporary research on the structure of the lymphoid system contributed to recognition of the microenvironment and secretory functions of the primary lymphoid organs. Determining the histology of organs, using electron microscopy and immunocytochemistry of cells, will catalyze interactions between morphologists and immunologists to create novel ideas and hypotheses, and will result in a more comprehensive understanding of avian immunology. This chapter is a comprehensive immune-morphological report on the primary and secondary lymphoid organs of birds.

A bird's eye view of enteric nervous system development: Lessons from the avian embryo

The avian embryo has been an important model system for studying enteric nervous system (ENS) development for over 50 y. Since the initial demonstration in chick embryos that the ENS is derived from the neural crest, investigators have used the avian model to reveal the cellular origins and migratory pathways of enteric neural crest-derived cells, with more recent work focusing on the molecular mechanisms regulating ENS development. Seminal contributions have been made in this field by researchers who have taken advantage of the strengths of the avian model system. These strengths include in vivo accessibility throughout development, ability to generate quail-chick chimeras, and the capacity to modulate gene expression in vivo in a spatially and temporally targeted manner. The recent availability of the chicken genome further enhances this model system, allowing investigators to combine classic embryologic methods with current genetic techniques. The strengths and versatility of the avian embryo continue to make it a valuable experimental system for studying the development of the ENS.

Origin of the bursal secretory dendritic cell

The origin of vimentin-positive secretory dendritic cells of the bursa of Fabricius was studied by chick–quail chimera, parabiosis and immunohistochemistry using species-specific monoclonal antibodies. Quail bursal primordia of different ages were transferred to coelomic cavity of 3-day-old chicken embryos and further incubated for 18 days. In transplanted quail bursas the secretory dendritic cells of chicken and quail origin were detected by double staining of vimentin plus 74.3 and vimentin plus QCPN monoclonal antibodies, respectively. In bursal primordia of 5- and 6-day-old quail embryos both dendritic cells and B cells were of host, i.e. chicken origin. Mixed dendritic cell population of quail and chick origin emerged in chimeric birds of 6.5 days of age. In quail embryos transplanted at 7 and 8 days of age both dendritic cells and B cells were mixed i.e. of chicken and quail origin. Bursal secretory dendritic cells and medullary epithelial cells create “dendro-epithelial tissue” to receive pre-B cells. Colonization of dendro-epithelial tissue by pre-B cells initiates at day 7, thus the colonization of bursal anlage by blood-borne cells is a two-step process; entering of dendritic cells at day 6.5 is followed by that of B cells at day 7 and afterwards. It is discussed that bursal secretory dendritic cells and their product are key elements of bursal function therefore the mammalian bursa equivalent organ might be represented by a cell, which is analogous with the bursal secretory dendritic cell.

Peripheral blood fibrocytes contribute to the formation of the avian spleen

Chick–quail chimeric studies were made to determine the origin of the cells of splenic ellipsoid. The ellipsoid is formed by supporting and phagocytic cells, which are embedded in a well‐organized extracellular matrix. Splenic and bursal anlage of 6‐ to 6.5‐day‐old quail embryos were transplanted into the coelomic cavity of 3‐day‐old chick embryos and further incubated for 17 days. CD45+ chicken hemopoietic cells colonized both organs. They formed the cells of the ellipsoid and the periellipsoidal white pulp of the transplanted quail spleen. Chicken‐specific collagen III was produced only in the donor quail spleen, but not in the bursa of Fabricius. The CD45+/collagen I+/collagen III+ cells are probably identical with the mammalian peripheral blood fibrocytes and contribute to the formation of supporting cells, whereas the CD45+/74.2+ ellipsoid‐associated macrophages are of monocytic origin. We provide, for the first time, experimental evidence that peripheral blood fibrocytes exist in the avian species; they are present in the circulation of the chicken embryo and contribute to the organogenesis of the spleen. Developmental Dynamics 232:55–66, 2005.

Enteric neural crest-derived cells promote their migration by modifying their microenvironment through tenascin-C production

The enteric nervous system (ENS) is derived from vagal and sacral neural crest cells that migrate, proliferate, and differentiate into enteric neurons and glia within the gut wall. The mechanisms regulating enteric neural crest-derived cell (ENCC) migration are poorly characterized despite the importance of this process in gut formation and function. Characterization of genes involved in ENCC migration is essential to understand ENS development and could provide targets for treatment of human ENS disorders. We identified the extracellular matrix glycoprotein tenascin-C (TNC) as an important regulator of ENCC development. We find TNC dynamically expressed during avian gut development. It is absent from the cecal region just prior to ENCC arrival, but becomes strongly expressed around ENCCs as they enter the ceca and hindgut. In aganglionic hindguts, TNC expression is strong throughout the outer mesenchyme, but is absent from the submucosal region, supporting the presence of both ENCC-dependent and independent expression within the gut wall. Using rat-chick coelomic grafts, neural tube cultures, and gut explants, we show that ENCCs produce TNC and that this ECM protein promotes their migration. Interestingly, only vagal neural crest-derived ENCCs express TNC, whereas sacral neural crest-derived cells do not. These results demonstrate that vagal crest-derived ENCCs actively modify their microenvironment through TNC expression and thereby help to regulate their own migration.

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. Anat Rec 262:279–292, 2001.

Gdnf is mitogenic, neurotrophic, and chemoattractive to enteric neural crest cells in the embryonic colon

Glial‐derived neurotrophic factor (Gdnf) is required for morphogenesis of the enteric nervous system (ENS) and it has been shown to regulate proliferation, differentiation, and survival of cultured enteric neural crest–derived cells (ENCCs). The goal of this study was to investigate its in vivo role in the colon, the site most commonly affected by intestinal neuropathies such as Hirschsprungs disease. Gdnf activity was modulated in ovo in the distal gut of avian embryos using targeted retrovirus‐mediated gene overexpression and retroviral vector‐based gene silencing. We find that Gdnf has a pleiotropic effect on colonic ENCCs, promoting proliferation, inducing neuronal differentiation, and acting as a chemoattractant. Down‐regulating Gdnf similarly induces premature neuronal differentiation, but also inhibits ENCC proliferation, leading to distal colorectal aganglionosis with severe proximal hypoganglionosis. These results indicate an important role for Gdnf signaling in colonic ENS formation and emphasize the critical balance between proliferation and differentiation in the developing ENS. Developmental Dynamics 240:1402–1411, 2011.

Identification of the Avian B-Cell-Specific Bu-1 Alloantigen by a Novel Monoclonal Antibody

A panel of monoclonal antibodies was generated against the guinea fowls bursal cells. One of the antibodies, designated BoA1, recognized both cortical and medullary B cells of bursal follicles and B cell dependent regions of peripheral lymphoid organs, like germinal centers and splenic periellipsoidal regions. The staining pattern of this monoclonal antibody is similar to other antibodies (L22, 11G2, AV20), which also identify the Bu-1 antigens. Under reducing conditions, the molecular weight of the BoA1 antigen is 70 to 73 kDa, and after immunoprecipitation it proved to be identical with the antigen recognized by the AV20 antibody. It is unique for this novel monoclonal antibody that it shows wide range cross-reactivity with different avian species, like chicken, quail, guinea fowl, and turkey. Therefore, this Bu-1-specific monoclonal antibody could be a versatile tool for studying the B cell development in different domesticated birds.

Development of the Avian Immune System

The avian embryo provides several advantages for studies on development of the immune system. These include the existence of a clear demarcation between the B and T cell systems, with each population differentiating in a specialized primary lymphoid organ—T cells in the thymus and B cells in the bursa of Fabricius. In addition, there is an availability of large numbers of embryos at precise stages of development. Because of its importance to the poultry industry, much research on the avian system has used the domestic chicken; this has been helped by the ready availability of different congenic and inbred lines, genetic markers and monoclonal antibodies (mAb)—the essential tools for studying the development of the immune system. The quail–chick chimeras have also proved to be an especially informative model, particularly for studying the emergence of hematopoietic stem cells (HSC) and their migration to the primary lymphoid organs during embryogenesis.

Experimental evidence for the ectodermal origin of the epithelial anlage of the chicken bursa of Fabricius

The bursa of Fabricius (BF) is a central lymphoid organ of birds responsible for B-cell maturation within bursal follicles of epithelial origin. Despite the fundamental importance of the BF to the birth of B lymphocytes in the immune system, the embryological origin of the epithelial component of the BF remains unknown. The BF arises in the tail bud, caudal to the cloaca and in close association with the cloacal membrane, where the anal invagination (anal sinus) of ectoderm and the caudal endodermal wall of the cloaca are juxtaposed. Serial semi-thin sections of the tail bud show that the anal sinus gradually transforms into the bursal duct and proctodeum, which joins the distal part of the cloaca during late embryogenesis. These anatomical findings raise the possibility that the ectoderm may contribute to the epithelial anlage of the BF. The expression of sonic hedgehog and its receptor in the embryonic gut, but not in the BF, further supports an ectodermal origin for the bursal rudiment. Using chick-quail chimeras, quail tail bud ectoderm was homotopically transplanted into ectoderm-ablated chick, resulting in quail-derived bursal follicle formation. Chimeric bursal anlagen were generated in vitro by recombining chick bursal mesenchyme with quail ectoderm or endoderm and grafting the recombination into the chick coelomic cavity. After hematopoietic cell colonization, bursal follicles formed only in grafts containing BF mesenchyme and tail bud ectoderm. These results strongly support the central role of the ectoderm in the development of the bursal epithelium and hence in the maturation of B lymphocytes.