Antibodies, immunoglobulin genes and the bursa of Fabricius in chicken B cell development

The bursa of Fabricius is critical for the normal development of B lymphocytes in birds. It is productively colonized during embryonic life by a limited number of B cell precursors that have undergone the immunoglobulin gene rearrangements required for expression of cell surface immunoglobulin. Immunoglobulin gene rearrangement occurs in the absence of terminal deoxynucleotidyl transferase and generates minimal antibody diversity. In addition, observations that immunoglobulin heavy and light chain variable gene rearrangement occur at the same time and that allelic exclusion of immunoglobulin expression is regulated at the level of variable region gene rearrangement provide a striking contrast to rodent and primate models of immunoglobulin gene assembly. Following productive colonization of the bursa, developing B cells undergo rapid proliferation and the immunoglobulin V region genes that generate the specificity of the B cell surface immunoglobulin receptor undergo diversification. Immunoglobulin diversity in birds is generated by somatic gene conversion events in which sequences derived from upstream families of pseudogenes replace homologous sequences in unique and functionally rearranged immunoglobulin heavy and light chain variable region genes. This mechanism is distinct from and much more efficient than mechanisms of antibody diversification seen in rodents and primates. While the bursal microenvironment is not required for immunoglobulin gene rearrangement and expression, it is essential for the generation of antibody diversity by gene conversion. Following hatch, gut derived antigens are taken up by the bursa. While bursal development prior to hatch occurs in the absence of exogenous antigen, chicken B cell development after hatch may therefore be influenced by the presence of environmental antigen. This review focuses on the differences between B cell development in the chicken as compared to rodent and primate models.     

The occurrence of spontaneous rosette-forming cells in the chicken bursa of Fabricius and spleen

The bursa of Fabricius of non-immunized chickens contained a significant number of cells forming rosettes (RFC) with sheep red blood cells. A considerably smaller number was found in the spleen. Spontaneous RFC may develop in the bursa and emigrate to peripheral lymphoid tissue.     

Selection for B cells with productive IgL gene rearrangements occurs in the bursa of Fabricius during chicken embryonic development

The vast majority of immunoglobulin-expressing mature chicken B lymphocytes contain one functionally rearranged and one unrearranged allele of the immunoglobulin light chain (IgL) gene. Therefore, nearly all IgL V-J rearrangements present in mature chickens are in-frame. In contrast, the Ig genes of mature mammalian B cells contain a high proportion of out-of-frame V-J joints. To investigate the basis for this difference, gene rearrangement at the chicken IgL locus was characterized during embryonic development and in mature B-cell lines. Joining of the single functional variable (VL) segment with the single joining (JL) segment occurs in cells in multiple tissues during a transient period of chicken embryogenesis. Only one-third of the V-J joints cloned from days 10-12 of development are in-frame. An increasing proportion of in-frame V-J joints is observed within the bursa of Fabricius at successively later stages of development. Our data suggest that the bursa of Fabricius serves during embryonic development as a site of selective amplification of cells that have undergone productive V-J joining, such that nearly all V-J joints present in postembryonic B cells are in-frame. The high frequency of rearranged alleles joined in-frame that is found in posthatching bursal cells and mature B-cell lines appears to result from a low frequency with which cells undergo IgL rearrangement at both alleles, rather than from an increase in the precision of V-J joining in avian species.     

Ontogeny of spontaneous rosette-forming cells in the chicken. With special reference to the bursa of Fabricius.

Cells forming rosettes (RFC) with sheep erythrocytes (SRBC) and rabbit erythrocytes (RRBC) were studied during the ontogeny of the bursa of Fabricius of the chicken. The frequency of SRBC-RFC was low but significant in the bursa of 15-day-old embryos, and increased therafter in an approximately linear fashion with the age of the bursa donor until at least in 43-day-old chickens. The total number of SRBC-RFC per bursa increased throughout the ontogeny with no evidence for any abrupt increase after hatch. The mean number of SRBC bound per RFC also increased after hatch. In contrast the frequency of bursal RRBC-RFC was high in 15-day-old embryos, significantly decreased on embryonic day 18, remained low until at least 7 days after hatch and then increased significantly. The total number of RRBC-RFC per bursa in contrast remained relatively constant during the embryonic period and the first week after hatch and thereafter increased markedly. No change in the mean number of RRBC bound per RFC could be demonstrated during the ontogeny. RRBC-RFC were also frequent in the yolk sac, spleen, and thymus of 15-day-old embryos.     

B cell emigration directly from the cortex of lymphoid follicles in the bursa of Fabricius

In the peripheral blood (PBL) of juvenile chickens three populations of B cells have previously been distinguished based on life-span and origin of cells within each population. In this report we show that the largest PBL B cell subset, population 1 B cells, which are short-lived cells corresponding to about 60% of PBL B cells and the vast majority of bursal emigrants, exit from the bursa directly from the follicular cortex. This conclusion is based on the specific labeling of rapidly dividing cortical lymphocytes with bromodeoxyuridine, followed by their detection in the periphery prior to the appearance of bromodeoxyuridine labeled cells in the bursa medulla. Furthermore, the rate of emigration of cortical lymphocytes, 1.00 ± 0.1% of PBL B cells per hour, is indistinguishable from the emigration rate of B cells from the bursa as a whole. The anatomical organization of the bursa has evolved to focus gut-derived antigens from the bursal lumen into the lymphoid follicles. The emigration of cortical bursal cells is discussed in relation to the exposure of bursal lymphocytes to extrinsic antigen.     

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.     

Nurse cells of the bursa of Fabricius: Do they exist?

Cell-cell interactions in B lymphocyte development have so far been incompletely characterized, mostly due to lack of a special organ for B cell maturation in the mammalian species. Certain well-known lymphostromal interactions in the thymus have raised the question whether similar interactions with nurse cells would also operate in the development of B cells. We have tested this hypothesis in the chicken bursa of Fabricius, an organ specific for the B cell maturation. To identify possible nurse cells, with viable lymphocytes enclosed, the cells in the bursa of Fabricius were dispersed with collagenase and trypsin. Light and electron microscopic examination of bursa cell suspensions showed four types of aggregates, identified by low magnification light microscopy as potential nurse cell-like complexes. Electron microscopy revealed that all aggregates consisted of epithelial cells, and complexes of epithelial cells with lymphocytes enclosed were not observed. These findings indicate that interactions similar to those seen in the avian and mammalian thymus between epithelial nurse cells and T lymphocytes are not a part of the avian B cell differentiation process.     

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.     

Diverse expression of the K-1 antigen by cortico-medullary and reticular epithelial cells of the bursa of Fabricius in chicken and guinea fowl

The immunocytochemical study of the K-1 monoclonal antibody indicates that the epithelial components of the bursa of Fabricius of the chicken and guinea fowl express the K-1 positive molecule. During embryogenesis, the K-1 antigen expression appears together with the bud-formation. As the number of B cells increases in the developing follicle, the K-1 expression gradually diminishes in the medullary reticular epithelial cells and completely ceases by hatching, which suggests that the molecule is developmentally regulated. After hatching, the expression of the molecule is restricted to the sealing off zone of the lymphoepithelial or medullary region of the follicle: i.e. to the cortico-medullary (CM) epithelial cells and the follicle associated epithelium (FAE) supporting cells in guinea fowl and to the latter ones in the chicken. The expression of the K-1 antigen by these epithelial components may support their structural identity. After hatching, the K-1 molecule is restricted to the CM epithelial cells and/or FAE supporting cells, which suggests that the function of the embryonic epithelial bud is taken over by the CM epithelial cells. The K-1 positive CM epithelial cells form arches, which encompass blast-like cells. The possible relationship of the CM epithelial cells and blast-like cells, which may represent the precursors of bursal secretory dendritic cells is discussed.     

Ontogenic restriction of colonization of the bursa of Fabricius

The capacity of hemopoietic precursor cells (HPC) to home to embryonic bursal and thymic grafts was investigated in embryonic and newly hatched chickens. Whereas thymic grafts developed normal histogenesis in both types of recipients, the bursal rudiment was colonized and developed in embryonic, but not in newly hatched hosts. In the latter, noncolonized bursal grafts developed neither lymphoid follicles nor granulopoiesis in the mesenchyme. These results are interpreted in terms of ontogenic “maturation” of the HPC which lose their homing potential towards the bursa while they preserve their thymic seeding capacity. This hypothesis is consistent with previously reported data which indicated cyclic continuous recruitment of the thymic lymphoid population, but restricted bursal colonization to a relatively brief period of embryonic life.     

In vitro organ culture of embryonic bursa of Fabricius

An in vitro method for organ culture of embryonic bursa of Fabricius is presented. It is shown that bursa cells proliferate in in vitro culture as evidenced by [3H]-thymidine incorporation. We assessed the expression of B-cell alloantigen (Bu-la and Bu-lb), class I (B-F) and class II (B-L) antigens of chicken major histocompatibility complex (MHC), and surface immunoglobulin (sIg) on cultured bursa cells using specific monoclonal antibodies (mAb). Cells from 13-day and 14-day embryonic bursae incubated in organ culture for 1 to 2 weeks developed characteristic patterns of surface phenotype observed in adult chicken except for B-F antigen, whose expression was much lower than in vivo. These results indicate that the maturation of bursa cells in organ culture follows the in vivo development, except for the expression of MHC class I antigens. Furthermore, we demonstrate the in vitro repopulation of bursae from cyclophosphamide(cy)-treated chickens by cells from Bu-l antigen disparate normal bursae.     

Follicular exclusion of retroviruses in the bursa of Fabricius

To gain insight into the regulation of retroviral infection at the cellular level, we analyzed the distribution of retroviral antigen and nucleic acid in the bursa of Fabricius of the parents and progeny of two highly inbred lines of chickens, one resistant and the other susceptible to infection. Line 15I5 chickens and line 7(2), which are C/C and C/A, respectively, and 15I5 x 7(2) F1 chickens were infected with either RAV-1 or RAV-49 avian leukosis virus (ALV). Most bursal follicles of F1 chickens infected with either virus contained a variable mixture of virus-positive and virus-negative cells and a few (1 to 20%) were void of detectable virus. However, in either parental line the respective virus was uniformly expressed among all follicles. The follicles which excluded virus in the F1 birds were indistinguishable from other infected follicles in the same bursa or in uninfected birds on the basis of histology or cellular antigen expression. It was concluded that virus susceptibility is most likely determined at the bursal stem cell level of differentiation, possibly by a process of allelic exclusion at the retroviral receptor locus.     

S-100 immunoreactive cells in the spleen and bursa of Fabricius of broiler chickens

The distribution of S-100 protein in spleens and bursae of Fabricius of broiler chickens with various diseases was investigated by an immunohistochemical method with antiserum to bovine S-100 protein. S-100 protein-positive cells were found in six cases of 34 chickens. In spleens, S-100 protein was detected in reticular cells in the ellipsoids of the sheathed arteries and eosinophilic granulocytes. Follicular dendritic cells in germinal centres were also S-100 protein-positive. In the bursa, reticular cells of the follicles and eosinophilic granulocytes in the follicular cortex and surface epithelium were positive for S-100 protein. The evidence suggests that S-100 protein may be involved in diseases of chickens.