Imre Oláh

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.

Characterization of chicken epidermal dendritic cells

It has been known for 15 years that the chicken epidermis contains ATPase+ and major histocompatibility complex class II‐positive (MHCII+) dendritic cells. These cells were designated as Langerhans cells but neither their detailed phenotype nor their function was further investigated. In the present paper we demonstrate a complete overlapping of ATPase, CD45 and vimentin staining in all dendritic cells of the chicken epidermis. The CD45+ ATPase+ vimentin+ dendritic cells could be divided into three subpopulations: an MHCII+ CD3– KUL01+ and 68.1+ (monocyte‐macrophage subpopulation markers) subpopulation, an MHCII– CD3– KUL01– and 68.1– subpopulation and an MHCII– CD3+ KUL01– and 68.1– subpopulation. The first population could be designated as chicken Langerhans cells. The last population represents CD4– CD8– T‐cell receptor‐αβ– and ‐γδ– natural killer cells with cytoplasmic CD3 positivity. The epidermal dendritic cells have a low proliferation rate as assessed by bromodeoxyuridine incorporation. Both in vivo and in vitro experiments showed that dendritic cells could be mobilized from the epidermis. Hapten treatment of epidermis resulted in the decrease of the frequency of epidermal dendritic cells and hapten‐loaded dendritic cells appeared in the dermis or in in vitro culture of isolated epidermis. Hapten‐positive cells were also found in the so‐called dermal lymphoid nodules. We suggest that these dermal nodules are responsible for some regional immunological functions similar to the mammalian lymph nodes.

Origin of follicular dendritic cell in the chicken spleen

The ellipsoid-associated cell (EAC) is a blood-borne phagocytic cell, residing in the antigen trapping zone of the chicken spleen. Binding and endocytosis of βGalactosidase (βGal) are independent from the Fc and complement receptors, because sulfated polysaccharides, in a concentration manner, inhibit the bacterial antigen uptake. The βGal-positive cells migrate to the periarterial lymphatic sheath (PALS), the preexisting germinal centers (GC), and form clusters with B- and T-cells. βGal, E5G12 double positive cells on the surface of the ellipsoid and in the PALS, GC and clusters prove that the EACs carry the enzyme. The EAC and the follicular dendritic cell (FDC) express, 68.2 and E5G12 and, 74.3 and E5G12, antigens, respectively. During migration the cessation of 68.2 and expression of 74.3 indicate the differentiation of EAC to FDC. By day 14 the clusters had disappeared, and in several GC the presence of double positive cells (74.3 and βGal; E5G12 and βGal) showed that the clusters had developed to GC. The presence of βGal+ cells in the PALS, where interdigitating dendritic cells (IDC) cooperate with the T-cells, suggests that in the spleen alternate routes exist for the EAC differentiation to FDC: EAC to FDC: βGal-loaded cells in the preexisting GC; and EAC through IDC to FDC: βGal+ EAC in the PALS and clusters. The EAC-FDC axis works exclusively inside the spleen; therefore; this system may be operated in pneumococcus infection.

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.

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.

The lymphoid substance of the chicken's Harderian gland is organized in two histologically distinct compartments

Light and electron microscopical investigations revealed that the lymphoid structure of the chicken Harderian gland is organized in different histological frameworks. In the head the surface epithelium of the central canal can be classified as a lymphoepithelial tissue which covers the dense lymphoid substance. It consists of small and medium‐sized lymphocytes, dendritic‐like cells, and occasional macrophages. High endothelial venules are associated with intense lymphocyte migration and homing that gives circumstantial evidence for a T‐dependent region, as found in a secondary lymphoid organ. The B‐dependent germinal centers are also common structural units of the head regions lymphoid substance. The body of the gland is loaded with plasma cells of different maturation stages. They immigrate into the epithelium of the central canal and produce IgM and IgA. Only a few scattered IgG producing plasma cells can be found in the gland of Harder. This plasmocytic region accounts for the immunosurveillance on the conjunctiva and in the upper respiratory tract through antibody production against bacterial or parasitic infections. In both the head and body regions of the gland, anti‐B‐L (anti‐Ia) antibody recognized scattered elongated cells which might represent dendritic cells. The immunological relationship between the two histologically different parts of the Harderian gland is unknown, but we speculate that the dense lymphoid tissue with high endothelial venule receives the blood‐borne, immunologically mature, but uncommitted B cells. By the influence of local antigen stimulus, these B cells transform to plasma cells which gradually appear in the body of the gland. The lymphoid structures of the head and the body fulfill the function of secondary and tertiary lymphoid organs, respectively.

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.

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.

The surface phenotype of swine blood and tissue eosinophil granulocytes

Cell surface antigens of swine eosinophil granulocytes were studied with flow cytometry and immunohistochemistry. The monoclonal antibody 335-2, specific for swine differentiation antigen swC1a, originally described to be present on swine T and myeloid cells, is able to distinguish swine eosinophils (swC1a negative) from neutrophils (swC1a positive). This monoclonal antibody (mAb) was used in two-colour fluorescence measurements in combination with anti-swine -CD2, -CD4, -CD8, -MHC class II, -LFA-1 or -swC3 mAbs. All of the blood eosinophils proved to be positive for LFA-1 and swC3, a common marker of swine monocytes, granulocytes and macrophages. However, they do not react with antibodies recognizing swine CD2, CD4, CD8 or MHC class II cell surface molecules. The reactivity pattern of tissue eosinophils with these mAbs was determined on cryostat sections of different tissues of swine. Tissue eosinophils were negative for swC1a, CD2, CD8, while all of them reacted with swC3. In contrast with blood eosinophils, 10-30% of tissue eosinophils were demonstrated to be negative for LFA-1. In some cases, a few tissue eosinophils were found to be stained weakly by antibodies to swine CD4 or MHC class II antigens.