Quail as the chimeric counterpart of the chicken: morphology and ontogeny of the bursa of Fabricius

The quail is the chimeric and parabiotic counterpart of the chicken, thus increasing the value of quail in the field of developmental biology. Quail bursa of Fabricius was studied by light microscopy, electron microscopy, and immunocytochemical methods. The basic cellular composition and structural framework are comparable with those of the chicken bursa. One of the major structural differences is the absence of the continuous cortico-medullary arch. In addition to the epithelial reticular cell the bursal secretory dendritic cell is the other medullary-specific bursal cell. The bursal secretory dendritic cell is a highly elongated cell which expresses vimentin intermediate filaments and produces secretory granules. The substance of the granules can be visualized by NIC2 monoclonal antibody, which was produced against guinea fowl bursal secretory dendritic cell. The released granular content appears on the lateral surface of the bursal secretory dendritic cell and is gradually solubilized. Thus, the NIC2-positive substance may occur in membrane-bound and solubilized forms in the isolated environment of the medulla. The bursal secretory dendritic cell establishes membrane contact areas with the B cells; therefore, they may influence B-cell maturation by cell contact and chemical (humoral) product. During embryogenesis bursal secretory dendritic cell precursors enter the epithelium and 1) induce epithelial bud formation, and 2) produce an NIC2-positive substance. Senescent bursal secretory dendritic cells can be phagocytic and migrate into the follicle-associated epithelium. This physiological turnover of the bursal secretory dendritic cell represents a novel pathway of macrophage formation from dendritic cells.     

Ontogeny of oestrogen-sensitive mesenchymal cells in the bursa of Fabricius of the chick embryo. An immunohistochemical study on progesterone receptor

The expression of progesterone receptor (PR) and its induction by oestradiol during the embryogenesis of the chick bursa of Fabricius (BF) were studied by immunohistochemistry using three different polyclonal antibodies to the chicken oviduct PR. Mesenchymal cells of the cloacal area surrounding the bursa primordium in controls (without exogenous oestrogen) express the PR between 9 and 11 days of incubation. In the same cells, PR was induced experimentally by oestradiol at 9 days. Mesenchymal cells in the bursa did not express PR after oestradiol treatment before the age of 11 days. The PR was not inducible in the bursal epithelium or in haemopoietic cells. None of the bursal cells expressed the PR to a detectable level during embryonic life without exogenous treatment. Some haemopoietic cells showed strong artefactual staining in their nuclei. It is concluded that (1) the embryonic bursa of Fabricius is a sex-steroid-sensitive organ, (2) exogenous oestradiol is able to induce progesterone receptor in the mesenchymal cells, but (3) the PR is not expressed without exogenous oestrogen. This indicates that the PR becomes oestrogen inducible well before it is naturally expressed during sexual maturation and that the level of endogenous oestrogen during embryonic life is not high enough to affect the bursa significantly.     

Selective expression of RAG-2 in chicken B cells undergoing immunoglobulin gene conversion

Chickens create their immunoglobulin (Ig) repertoires during B cell development in the bursa of Fabricius by intrachromosomal gene conversion. Recent evidence has suggested that Ig gene conversion may involve cis-acting DNA elements related to those involved in V(D)J recombination. Therefore, we have examined the potential role of the V(D)J recombination activating genes, RAG-1 and RAG-2, in regulating chicken Ig gene conversion. In contrast to the coexpression of RAG-1 and RAG-2 observed in mammalian B cells that undergo V(D)J recombination, chicken B cells isolated from the bursa of Fabricius express high levels of the RAG-2 mRNA but do not express RAG-1 mRNA. The developmental and phenotypic characteristics of the bursal lymphocytes and chicken B cell lines that express RAG-2 mRNA demonstrate that selective RAG-2 expression occurs specifically in B cells undergoing Ig diversification by gene conversion. These data suggest that RAG-2 plays a fundamental role in Ig-specific gene conversion.     

Expression and purification of chicken beta interferon and its antivirus immunological activity

Interferon beta (IFN-β) belongs to a class of natural proteins that can inhibit virus replication. In this paper, nucleic acid sequence encoding chicken interferon beta (chIFN-β) mature protein was cloned and highly expressed in Escherichia coli (E. coli) by 1mM IPTG induction. The expressed chIFN-β was about 20% of total bacterial protein. The recombinant protein in form of inclusion body was then solubilised, refolded and purified to a purity of greater than 95% by immobilized metal ion affinity chromatography (IMAC). This recombinant chIFN-β could inhibit vesicular stomatitis Indiana virus (VSV) and infectious bursal disease virus (IBDV) replication in vitro. Animal experiments showed that the recombinant chIFN-β could decrease pathological lesions caused by the IBDV in bursa of Fabricius. These results will provide useful information for further investigations on veterinary clinical applications and fundamental research.     

A model of bursal colonization

In the chicken, and perhaps in all birds, the development of the cells which form antibodies, the B-cells, differs substantially from that in mammals. In birds, committed B-cells colonize a specialized organ, the bursa of Fabricius, which consists of some 10(4) follicles. Diversification, i.e., the development of the antibody repertoire, takes place in bursal follicles by a process termed "gene conversion." The avian bursa is easily accessible experimentally, and in the chicken, it has been the subject of extensive research. As an aid to experimentation in this field, we present here a formal mathematical model of bursal development. Formulae are derived which allow one to estimate the number and sizes of B-cell clones in bursal follicles, and hence the size of the overall antibody repertoire. Particular attention is paid to the problem of estimating experimental errors.     

Immune complexes of E. coli antigens and maternal IgG in the bursa of Fabricius

The bursa of Fabricius of the chicken is known to be both a primary lymphoid organ and a secondary lymphoid tissue. Bursal follicles are equipped with antigen-trapping follicle-associated epithelium. However, bioactive antigens such as protein and bacteria have not been detected in the bursal parenchyma. By immunoperoxidase staining with a polyspecific antibody (Ab) against Escherichia coli, we detected aggregated E. coli antigens in the medulla of bursal follicles after hatching. The distribution of aggregated E. coli antigens is restricted to the medulla of bursal follicles. The antigens are not found in the spleen or the parenchyma of the caecal tonsil. The bursa is thus a trapping site for E. coli antigens from the external environment. Furthermore, two-color immunostaining clarified that these antigens form immune complexes with maternal IgG (MIgG) and are retained by reticular cells. Additionally, immune complexes in the bursa were shown to induce the rapid development of serum IgM Ab for indigenous E. coli. Our results suggest that immune complexes of MIgG and environmental antigens in the medulla of bursal follicles exert positive effects on B-cell differentiation in the bursa in situ.     

Establishment of an In Vitro System Representing the Chicken Gut-Associated Lymphoid Tissue

The bursa of Fabricius is critical for B cell development and differentiation in chick embryos. This study describes the production in vitro, from dissociated cell suspensions, of cellular agglomerates with functional similarities to the chicken bursa. Co-cultivation of epithelial and lymphoid cells obtained from embryos at the appropriate developmental stage regularly led to agglomerate formation within 48 hours. These agglomerates resembled bursal tissue in having lymphoid clusters overlaid by well organized epithelium. Whereas lymphocytes within agglomerates were predominantly Bu-1a⁺, a majority of those emigrating onto the supporting membrane were Bu-1a⁻ and IgM⁺. Both agglomerates and emigrant cells expressed activation-induced deaminase with levels increasing after 24 hours. Emigrating cells were actively proliferating at a rate in excess of both the starting cell population and the population of cells remaining in agglomerates. The potential usefulness of this system for investigating the response of bursal tissue to avian Newcastle disease virus (strain AF2240) was examined.     

Cell lineage segregation during bursa of Fabricius ontogeny

The population dynamics of myeloid and lymphoid lineages during bursa of Fabricius ontogeny were analyzed by immunofluorescence by using two monoclonal antibodies (mAb). CL-1 mAb reacts with all chicken hemopoietic cells, except mature erythrocytes. L22 mAb reacts with bursa and bursa-derived lymphocytes, with a minor subset of macrophages and with some cells of the thymic medulla. The staining of embryonic bursas by these antibodies helps to distinguish between two different lineages of hemopoietic cells: CL-1+/L22+ cells represent B lymphocytes and a minor subset of macrophages, while CL-1+/L22- cells correspond to most of the macrophages and to the granulocytes, which disappear at the end of the embryonic life. CL-1+/L22- as well as CL-1+/L22+ cells were first observed outside the bursal rudiment. This indicates that there is a pre-bursal segregation between these two hemopoietic lineages and that two different kinds of precursors colonize the bursal rudiment at about the same time (day 9 for CL-1+/L22- cells and days 9 or 10 for CL-1+/L22+ cells). Moreover our data show that the colonization of the bursal epithelium by hemopoietic precursors is a two-step phenomenon. The first cells which enter belong to the CL-1+/L22- lineage, express Ia-like antigens at a high level, are dendritic in morphology, and represent cells of the macrophage/dendritic cell lineage. They are responsible for the formation of the epithelial bud which are then colonized by a small number of lymphoid precursors which belong to the CL-1+/L22+ lineage. Quail-chick bursa grafting experiments were also performed and the grafts were examined for CL-1 (restricted to chicken hemopoietic cells) and L22 reactivity. These observations confirmed our previous findings about the kinetics of the colonization of bursal rudiment by hemopoietic precursors and give support for a pre-bursal segregation between two hemopoietic pathways.     

Characteristics of bursal T lymphocytes induced by infectious bursal disease virus

Infectious bursal disease virus (IBDV) is an avian lymphotropic virus that causes immunosuppression. When specific-pathogen-free chickens were exposed to a pathogenic strain of IBDV (IM), the virus rapidly destroyed B cells in the bursa of Fabricius. Extensive viral replication was accompanied by an infiltration of T cells in the bursa. We studied the characteristics of intrabursal T lymphocytes in IBDV-infected chickens and examined whether T cells were involved in virus clearance. Flow cytometric analysis of single-cell suspensions of the bursal tissue revealed that T cells were first detectable at 4 days postinoculation (p.i.). At 7 days p.i., 65% of bursal cells were T cells and 7% were B cells. After virus infection, the numbers of bursal T cells expressing activation markers Ia and CD25 were significantly increased (P<0.03). In addition, IBDV-induced bursal T cells produced elevated levels of interleukin-6-like factor and nitric oxide-inducing factor in vitro. Spleen and bursal cells of IBDV-infected chickens had upregulated gamma interferon gene expression in comparison with virus-free chickens. In IBDV-infected chickens, bursal T cells proliferated in vitro upon stimulation with purified IBDV in a dose-dependent manner (P<0.02), whereas virus-specific T-cell expansion was not detected in the spleen. Cyclosporin A treatment, which reduced the number of circulating T cells and compromised T-cell mitogenesis, increased viral burden in the bursae of IBDV-infected chickens. The results suggest that intrabursal T cells and T-cell-mediated responses may be important in viral clearance and promoting recovery from infection.     

The origin of IgG-containing cells in the bursa of Fabricius

The bursa of Fabricius of the chicken is known as a primary lymphoid organ for B-cell development. Morphologically, the origin of IgG-containing cells in the bursa has not been clear until now, because abundant maternal IgG (MIgG) is transported to the chick embryo and distributed to the bursal tissue around hatching. Thus, it has been difficult to find out whether these cells themselves biosynthesize IgG or if they acquire MIgG via attachment to their surface. Our present study employing in situ hybridization clarified that IgG-containing cells in the medulla of bursal follicles did not biosynthesize IgG. To study the role of MIgG in the development of those IgG-containing cells, MIgG-free chicks were established from surgically bursectomized hen (SBx-hen). We found that, on the one hand, deprivation of MIgG from chicks completely inhibited the development of IgG-containing cells in the medulla after hatching. On the other hand, administration of MIgG to MIgG-free chicks recovered the emergence of those cells. In addition, we observed that those cells did not bear a B-cell marker and possessed dendrites with aggregated IgG. These results demonstrate that IgG-containing cells in the medulla are reticular cells that capture aggregated MIgG. Moreover, we show that the isolation of the bursa from environmental stimuli by bursal duct ligation (BDL) suppressed the development of IgG-containing cells after hatching. Thus, it is implied that environmental stimulations play a key role in MIgG aggregations and dendritic distributions of aggregated MIgG in the medulla after hatching.     

Expression of caveolin‐1 in the interfollicular but not the follicle‐associated epithelial cells in the bursa of fabricius of chickens

The surface epithelium of the bursa of Fabricius consists of interfollicular (IFE) and follicle‐associated epithelium (FAE). The IFE comprises (i) cylindrical‐shaped secretory cells (SC) and (ii) cuboidal basal cells (BCs). The FAE provides histological and two‐way functional connections between the bursal lumen and medulla of the follicle. We used a carbon solution and anti‐caveolin‐1 (Cav‐1) to study the endocytic activity of FAE. Carbon particles entered the intercellular space of FAE, but the carbon particles were not internalized by the FAE cells. Cav‐1 was not detectable in the FAE cells or the medulla of the bursal follicle. The absence of Cav‐1 indicates that no caveolin‐mediated endocytosis occurs in the FAE cells, B cells, bursal secretory dendritic cells (BSDC), or reticular epithelial cells. Surprisingly, a significant number of Cav‐1 positive cells can be found among the SC, which are designated SC II. Cav‐1 negative cell are called SC I, and they produce mucin for lubricating the bursal lumen and duct. Occasionally, BCs also express Cav‐1, which suggests that BC is a precursor of a SC. Transmission electron microscopy confirmed the existence of type I and II SC. The SC II are highly polarized and have an extensive trans‐Golgi network that is rich in different granules and vesicles. Western blot analysis of bursa lysates revealed a 21–23 kDa compound (caveolin) and Filipin fluorescence histochemistry provided evidence for intracellular cholesterol. High amount of cholesterol in the feces shows the cholesterol efflux from SC II. The presence of Cav‐1 and cholesterol in SC II indicates, that the bursa is a complex organ in addition to possessing immunological function contributes to the cholesterol homeostasis in the chickens.     

Appearance of Extrachromosomal Circular DNA in Lymphocytes from Developing Chick Bursa

Lymphocytes were purified from the developing bursa of Fabricius of chick embryos and chickens and pressed with a mica sheet. Then the extruded DNA complexes were adsorbed to the mica and processed for electron microscopy. Circular DNA complexes were found at 40 to 60 copies in all bursal lymphocyte except 12-day-old embryonic bursas, which contained 150 copies. Circular DNA complexes of more than 1 μm (larger circular DNA) appeared in 12-day-old embryonic bursas at more than 20 copies per cell, and subsequently decreased in number to less than 10 copies per cell. These changes in the size distribution and copy number of circular DNA coincided with lymphocyte differentiation of the hemopoietic precursor cells in the bursa after seeding.     

Regulating properties of peptides of the thymus and bursa of Fabricius in immunodepression in birds

The intensity of nucleic acids and protein synthesis in the cells of the thymus and the bursa of Fabricius was studied in chickens against the background of an immunodepression induced by administration of hydrocortisone and cyclophosphamide. It was found out that hydrocortisone causes in chicken a marked lowering of the intensity of inclusion of 3H-thymidine, 3H-uridine and 14C-glycine in thymic cells, and cyclophosphamide--in the cells of the bursa of Fabricius. Under the conditions of selective immunodepression the preparations on the basis of the peptides of the thymus (thymalin) and of the bursa of Fabricius (bursilin) regulate the processes of nucleic acids and protein synthesis chiefly in the cells of organs which produce them.     

Effect of environmental antigens on the Ig diversification and the selection of productive V-J joints in the bursa

In chickens, a single set of unique functional segments of both Ig H and L chain genes is rearranged during early embryogenesis to generate a pool of B cell progenitors that will be diversified in the bursa by gene conversion, forming the preimmune repertoire. After hatching, bursal cells are exposed to environmental Ags in the bursal lumen. We prepared B cells from each single bursal follicle and used PCR-directed Ig L chain gene analysis to study the differentiation of B cells and the effect of antigenic stimulation from the bursal lumen on the neonatal chicken B cell repertoire formation. Selective amplification of B cell clones with a productive V-J joint was observed during the late embryonic stage, possibly by the interaction with ligands expressed on the bursal stroma and further accelerated in the neonatal chicken. Administration of the artificial Ags into the bursal lumen before the isolation of bursa by bursal duct ligation in the embryo caused a significant increase in lymphocytes with a productive V-J joint in the neonatal chicken bursa compared with the isolated bursa. Intra- and interclonal diversity of a complementarity-determining region measured by an evolutionary distance increased during bursal development. Clonal diversification did not require stimulation by artificial Ags from the bursal lumen. Thus, the preimmune repertoire in the bursa is generated by gene conversion during Ag-independent B cell proliferation, and antigenic stimulation from the bursal epithelium to bursal B cells plays roles in the selection of clones with a productive V-J joint.     

Identification of B-L antigens on reticular epithelial cells of the bursa of Fabricius

We have established two monoclonal antibodies against B-L antigens (chicken Ia-like antigens). The specificity of the antibodies for B-L antigens was determined by two criteria, the cellular expression and the molecular structure of antigens with which they reacted. They reacted with antigens expressed on bursacytes, Con A-blast thymocytes, macrophages, and MDCC MSB1, but not with thymocytes and erythrocytes. In molecular basis, they recognized 64,000 dalton glycoprotein consisting of two polypeptides, 35,000 and 32,000 dalton, which bound non-covalently. To investigate the distribution of B-L antigens on non-lymphoid cells of the bursa of Fabricius, which were thought to play important roles in the differentiation of B cells, anti-B-L antigen and anti-chicken immunoglobulin (Ig) monoclonal antibodies were used. B-L antigen-positive cells were detected in both cortical and medullary areas, whereas Ig-positive lymphoid cells were confined to the medullary areas of normal chicken bursal follicles. In the bursal follicles of cyclophosphamide (CY)-treated chickens, lymphoid cells were depleted but epithelial cells remained intact. And B-L antigen-positive but Ig-negative cells were easily detected in the medullary areas of almost all follicles. These cells were identified to be reticular epithelial cells (REp cells) from the result of their keratin expression.     

Ontogeny of lymphocytes expressing J chain in chickens

The ontogeny of chicken lymphocytes expressing J chain (LEJ) was investigated in the embryonic bursa of Fabricius, the spleen, and the thymus. Simultaneous appearance of LEJ was detected in the bursa and spleen on Day 14 of incubation. These cells were detected later in the thymus. The LEJ were found to increase rapidly in the spleen from the 19th to 20th incubation day. In adult chickens, the highest percentage of LEJ was also found in the spleen. These cells were seen in the thymus at a lower frequency. Intermediate numbers were found in bursal and peripheral blood lymphocytes. The frequencies of the LEJ were similar to those of lymphocytes positive for cytoplasmic immunoglobulins (Ig) IgA and IgM, but were not related to the number of lymphocytes expressing surface Ig. It is possible to consider that the suitable site for LEJ is the spleen, on the basis of the rapid increase in the number of LEJ just before hatching and from the fact that the highest value is found in adult chickens. Furthermore, LEJ may participate in secretion of IgA or IgM but not be associated with the expression of surface Ig.     

山羊羔淋巴集结的研究

对不同发育时期山羊羔淋巴集结、简称ＰＰ的显微和亚显微结构的观察显示羊ＰＰ的发生和组织学特征与鸟类法氏囊极为相似。同时,羊羔ＰＰ提取液可提高仔兔和仔鸡血清抗体的滴度,促进淋巴组织的发育。实验结果表明山羊ＰＰ是Ｂ淋巴细胞发生的重要部位,山羊ＰＰ中含有类似法氏囊素的物质。     

The effect of alterations in myc gene expression on B cell development in the bursa of Fabricius

Infection of 18-day embryonic bursal lymphocytes with a v-myc-containing retrovirus leads directly to a polyclonal proliferation of surface immunoglobulin-positive (slg+) cells in the bursa of Fabricius detected four weeks after hatching. These v-myc-expressing bursal cells repopulate the follicles of chemically ablated bursae more efficiently than total normal 18-day embryonic bursal cells. In contrast, comparable normal bursal cells lose the ability to repopulate follicles by four weeks. Bursal lymphocytes expressing either a retroviral v-myc or a c-myc gene deregulated by adjacent retroviral integration retain the ability of embryonic bursal lymphocytes to diversify their immunoglobulin light chain genes. These results suggest that retroviral deregulation of myc expression during avian B cell development induces outgrowth of a population of cells with the cardinal phenotypic characteristics of bursal stem cells.