MERIAL AVIAN SCIENCE REVIEW THE BURSA OF FABRICIUS AND ITS ESSENTIAL ROLE IN B-CELL DEVELOPMENT AND ANTIBODY PRODUCTION. BY PROF. BERND KASPERS PROFESSOR OF ANIMAL PHYSIOLOGY UNIVERSITY OF MUNICH - GERMANY revue_merial_2.indd 1
Birds are constantly challenged by a diverse set of pathogens such as viruses, bacteria, protozoa, helminthes and ecto-parasites (Figure 1). Figure 1. Viruses Bacteria Innate immune response Macrophages, Heterophils Interferon, Complement, Lysozyme Rapid response, no memory Fungi* Parasites Adaptive immune response B-lymphocytes, T-lymphocytes Immunoglobulins, Cytokines Delayed response, memory * Courtesy of Prof Jacques Guillot - Veterinary School of Maisons-Alfort These micro-organisms have developed unique and B- and T-lymphocytes which share a unique divers strategies to invade the host, to replicate and property: the ability to memorize previous contact to evade the defense system. On the other side, with pathogens and to recall this encounter during the immune system has co-evolved as a complex reinfection, thus mounting a rapid and highly efficient organ with multiple defense mechanisms to immune response. This short paper will focus on the respond appropriately to the plethora of pathogens. B-lymphocyte system. It is composed of a rapid and a delayed response First, a brief review of the structure and function system, called the innate and the adaptive immune of antibodies, the primary secretory product of system, respectively. B-cells, will be presented. Next, the development Frequently, the innate immune system is sufficient of B-cells during embryogenesis and the role of the to control infections but, if this is not the case, the bursa of Fabricius will be discussed. Finally, it will adaptive immune system will become activated. be explained how B-cells are activated to generate It should be noted that both systems closely interact; antibodies and how immunological memory is examples of this cooperation will be presented later. achieved. The role of T-lymphocytes will only be The adaptive immune system is composed of discussed in the context of T-cell help for B-cells. revue_merial_2.indd 2
THE ANTIBODY Antibodies are proteins which are produced by degree as a secreted protein on mucosal surfaces. B-cells and plasma cells and secreted into the tissues During the course of a primary infection, and in and blood (1). Because of their globular structure response to secondary infections, IgY becomes the they have also been named immunoglobulins. most prominent immunoglobulin. IgY is a monomer In chickens three types of immunoglobulins are and secreted at high concentrations into the blood present, which are IgM, IgY and IgA (figure 2). Birds (7-12 mg/ml), but it is found at comparatively low lack a homologue of the mammalian IgE molecule. amounts on mucosal surfaces. The basic structure of all immunoglobulins is During egg yolk formation, IgY is actively transported Y-shaped and composed of two smaller (light chain) from the hens blood into the yolk. As the developing and two larger (heavy chain) proteins linked together embryo absorbs the yolk content, IgY is transferred by disulfide bonds (Figure 2A). On one end the into the embryonic circulation to protect the chicken immunoglobulin molecule can bind antigen while during the first critical time after hatch. In contrast the other end mediates important effector functions to IgM and IgY, IgA is found in small quantities in once the antigen is bound. The first immunoglobulin the blood. Nevertheless, IgA is the most abundant produced during an immune response is IgM immunoglobulin in the body, since it is produced (Figure 2B). In this molecule five of the basic Y-shaped in very large quantities by B-cells of the mucosal units are fused to form a pentamer, which allows tissues and is directly secreted as a dimer or trimer the molecule to bind multiple antigens at the same onto the mucosal surface to prevent microorganisms time. IgM is largely found in the blood and to a minor from entering. Figure 2. revue_merial_2.indd 3
Antibodies act in many different ways to control infections. However, their activity is largely restricted to the extracellular space. Intracellular pathogens are not well controlled by antibodies; examples are viruses while replicating inside the cell and intracellular bacteria such as Salmonella, Listeria or Mycobacteria. However, in the extra-cellular space and on mucosal surfaces, antibodies can firmly bind to soluble molecules or structural components of micro-organisms. Binding of antibodies to bacterial or fungal toxins, can neutralize their toxic activity which is best known from anti-tetanus toxin antibodies induced during vaccination (Figure 2C). In the case of viruses, antibodies can bind to viral antigens during viremia (IgY) or on mucosal surfaces (IgA) and prevent infection of cells through the inhibition of virus-cell interaction (Figure 2D). For example, antibodies directed against the hemagglutinin of influenza virus act in this way. Binding of antibodies to bacteria can mediate bacterial killing through different pathways. Coating of bacteria with antibodies is called opsonization and will lead to complement activation and subsequently to bacterial lysis by the complement components (Figure 2E). Furthermore, coated bacteria are recognized by heterophils and macrophages which will phagocytose and kill the pathogens. To identify opsonized micro-organisms, phagocytes carry on their cell surface receptors which bind to the antibodies coating the surface of the pathogen (Figure 2F). None of these mechanisms can be compensated effectively in antibody deficient animals. Therefore, antibody deficiency is a live threatening condition. revue_merial_2.indd 4
B-CELL DEVELOPMENT I N T H E B U R S A O F FA B R I C I U S during the B-cells leave the bursa and populate the embryogenesis and the first few weeks after peripheral tissues. This event starts around hatch. The central organ in this process is hatch and is believed to continue until the the bursa of Fabricius which is connected by bursa disappears at sexual maturation of an open duct to the proctodeum (figure 3). the birds. However, bursal development is It provides the environment for immature not accomplished at hatch. During the first B-cells to differentiate into fully functional two weeks after hatch the follicles undergo a antibody (2). structural transformation with the development Consequently, surgical removal of the bursa of a follicular cortex and a medulla (Figure 3.3). or destruction of the organ by chemicals, It is believed that the medulla functions as a toxins or viruses, leads to severe immuno- secondary lymphoid organ very much like the suppression and high susceptibility of the spleen while the cortex generates new B-cells birds to pathogenic challenge, as well known which continue to populate the peripheral by avian veterinarians. Between embryonic day lymphoid organs (Figure 3.4). Surgical removal (ED) 8 and 15, Pre-bursal B-cell precursors of the bursa has shown that this organ is of (B-cell stem cells ) migrate from the embryonic critical importance until chickens are at least at spleen and bone marrow into the bursa (Figure the age of 4-5 weeks. At that time a population 3.1). Here they receive signals from the bursal of B-cells starts to appear in the spleen that tissue which activate the maturation program. can maintain the B-cell system of chickens in The nature of these signals is still largely which the bursa was removed (4). However, unknown. However, the events taking place the bursa is still required during the following in the developing B-cells in response to these weeks, to guarantee a fully functional B-cell signals, have been studied intensively. Initially system. The B-cell system producing develops B-lymphocytes the bursa is populated by a few thousand pre-bursal progenitors which, as a first step, start to proliferate and to form aggregates of B-cells called bursal follicles (Figure 3.2). It has Figure 3. been estimated that a mature bursa contains up to 12.000 follicles with roughly 2 x 105 B-cells per follicle. During B-cell proliferation, a second important process is initiated, which ultimately leads to the formation of billions of B-lymphocytes, each of them producing a single unique antibody molecule with a unique binding specificity. The molecular mechanism of this process is well known and has been described in detail (3). Once fully matured, revue_merial_2.indd 5
B-CELL FUNCTION IN S E C O N D A R Y LY M P H O I D T I S S U E S B-cells leaving the bursa colonize secondary lymphatic structures, in particular the spleen, the caecal tonsils (CT) in the gut, the bronchus associated lymphoid tissue in the lung (BALT) and lymphoid follicles in the mucosal tissues. Here the cells get in contact with pathogens or with antigen delivered by vaccination. As outlined before, B-cell development in the bursa leads to billions of cells each of them producing an antibody with a different antigen binding side. This immunoglobulin molecule is anchored in the cell membrane, with its antigen binding site facing towards the environment (figure 4; upper part). It is an IgM type immunoglobulin expressed as a monomer and is referred to as the B-cell antigen receptor or BCR. Binding of the proper antigen (an antigen that fits at least decently well) to the BCR, will activate the B-cell and induce its proliferation and differentiation to an antibody secreting plasma cell. During this process, plasma cells lose their BCR on the cell surface but start to synthesize large amounts of their immunoglobulin molecule and secret it into the tissues and blood. This activation process requires 5-6 days, explaining why the appearance of the first antigen specific IgM antibodies is observed nearly one week after vaccination. A characteristic feature of the early IgM antibodies, is their low antigen affinity which results from an imperfect fit between the antigen and the binding site of the IgM molecule. This disadvantage is compensated by the pentameric structure of the secreted IgM, which allows simultaneous binding at multiple sites thus increasing the overall binding strength. Figure 4. Two features of the antibody response have fascinated immunologists for a long time. The first one is the appearance of IgY or IgA antibodies in the course of the response to an infection, a process that is called immunoglobulin class switch. The second one is the production of antibodies with increasingly stronger binding to the antigen, called affinity maturation. Both events take place in a well-known structure in lymphoid tissues, the germinal center (GC). In birds they are round structures found in the spleen, the caecal tonsils and the BALT (Figure 4: photo). After the first encounter of an antigen, some of the proliferating cells will initiate the formation of GCs. revue_merial_2.indd 6
During this process the cells will continue to Consequently, absence of T-helper cells, or divide and at the same time will start to mutate functional the immunoglobulin gene (figure 4; lower part). immune deficiency in the B-cell system. insufficiency, will cause severe These mutations will take place in the antigen binding site and change it in such a way that While affinity maturation and class switching the binding strength is either unchanged or lead to improved protection, the formation of worsened or, preferably, improved. Only those memory cells will help animals to respond very B-cells immunoglobulin rapidly with the secretion of high affinity IgY molecule will continue to proliferate and, in a or IgA antibodies to reinfection. Memory cells next step, will receive signals to change from generated in GCs during the primary infection IgM to IgY or IgA (class switch). (figure 4), can live for months to years. In mice Finally, these cells will differentiate to plasma and man these cells survive in the bone marrow. cells and start to secret large amounts of Their location in birds is largely unknown but their immunoglobulin. The final outcome of their existence is absolutely certain. Induction of the process is the generation of antibodies memory cells is the primary aim of vaccination. which can grab the antigen more strongly Primary vaccination will lead to the generation and consequently eliminate the pathogen most of memory cells which become reactivated efficiently. This is an important mechanism as during booster vaccination or in case of illustrated by the highly increased susceptibility infection by the same agent. This secondary to infections in patients with a failure in the response generates large numbers of B-cells class switching process. Such defects, albeit derived from the memory B-cell pool, which rare, have been described in man and mice produce even larger amounts of antibodies with but also in chicken lines. It should be noted further increased binding strength. In addition, that this process does not take place in the new memory cells are formed, to be available absence of T-cells. Critical signals for affinity for a subsequent challenge by the respective maturation and class switching are provided pathogen. with an improved by the so called T-helper cells. References 1. Härtle S, Magor K.E., Göbel, T.W., Davison F. Kaspers B. Structure and evolution of avian immunoglobulins. pp 103-120; In: Avian Immunology, Schat K.A., Kaspers B., Kaiser P. 2 nd edition, Elsevier 2013. 2. Oláh I, Nagy N. Retrospection to discovery of bursal function and recognition of avian dendritic cells; past and present. Dev Comp Immunol. 2013, 41(3):310-5 3. Ratcliffe M.J.H., Härtle, S. B cells, the bursa of Fabricius and the generation of antibody repertoires. pp 65-90; In: Avian Immunology, Schat K.A., Kaspers B., Kaiser P. 2 nd edition, Elsevier 2013. 4. Toivanen P, Toivanen A. Bursal and postbursal stem cells in chicken. Functional characteristics. Eur J Immunol. 1973, 3(9):585-95 revue_merial_2.indd 7
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