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.     

A novel method to bursectomize avian embryos and obtain quail----chick bursal chimeras. II. Immune response of bursectomized chicks and chimeras and post-natal rejection of the grafted quail bursas

Two methods to bursectomize chick embryos before hemopoietic cell seeding of the bursa of Fabricius were compared in this work: section of the tail region at E3 including the presumptive bursal territory, and selective removal of the bursa at E5. Hatching ability is better with the former method, but survival rate and effectiveness of bursectomy are favored with the second, novel technique. Moreover, selective removal of the bursa at E5 can be followed by in situ engraftment of a quail bursa and construction of quail-chick bursal chimeras. The immune response of bursaless birds and bursal chimeras has been studied. Total absence of the bursa does not prevent a few B cells from differentiating and nonspecific Ig (IgM and/or IgG) from being secreted. As reported previously, bursaless birds, however, are unable to mount an immune response by producing specific antibodies. This immune function is restored by the graft of a quail bursa. The microenvironment of the bursa, although heterospecific, allows the expansion of the B cell population and generates the repertoire of the B cell antigen receptors. This process takes place during late embryonic and early postnatal life because the grafted quail bursal stroma is subjected to immune rejection from 2 to 3 wk after birth in all chimeras, which are, however, perfectly immunocompetent.     

Sex steroid sensitivity of developing bursa of Fabricius

Sex steroid sensitivity of the bursa of Fabricius (BF) was studied from the early embryonic time until its regression. Expression of progesterone receptor (PR) served as a dual marker: first, as a marker for progesterone sensitivity and second, as a marker for estrogen action, since it is an estrogen-induced protein. The progesterone binding molecule in the bursa was characterized by different chromatography methods and by steroid binding studies. We showed that it fulfils the criteria of a progesterone receptor by binding, structural and immunological properties. With immunohistochemistry and with the combined techniques of immunohistochemistry and autoradiography we demonstrated two cell types which express the PR: smooth muscle cells surrounding the BF and stromal cells located under the bursal epithelium and between the lymphoid follicles. The epithelium and the cells inside the lymphoid follicles were negative. Using immunoelectron microscopy the PR-expressing stromal cells were shown to be fibroblasts. The cloacal mesenchyme, from which the BF develops, was shown to be sensitive to exogenous estrogen very early during the embryonic time. The mesenchyme around and inside the developing BF reached estrogen sensitivity a few days later. The estrogen-sensitive mesenchymal cells were first seen surrounding the bursal primordium and later in the center of the plicae. During a natural sexual maturation without exogenous estradiol an expression of the PR was detected much later, at the age of 10-12 weeks after hatching. This expression correlates with the onset of the bursal regression and with the increase of the sex steroid levels in the blood. In the oviduct stroma PR was undetectable before the onset of sexual maturation. In the oviduct stroma PR becomes detectable a few weeks earlier than in the bursa.     

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.     

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.     

A novel method to bursectomize avian embryos and obtain quail----chick bursal chimeras. I. Immunocytochemical analysis of such chimeras by using species-specific monoclonal antibodies

Chick embryos were bursectomized at 5 days of incubation according to a novel surgical technique described in this article. This method yields birds that are able to hatch and are devoid of the physiologic deficiencies resulting from the previously used method, which involved resection of the cloacal and posterior embryonic region. The bursectomized embryos were grafted in situ with a quail bursa of the same age, which thereafter became chimeric through chick host hemopoietic cell invasion. By means of species-specific antibodies, the chimeric condition revealed 1) that the bursal epithelium expresses a unique antigenic determinant (MB1 determinant), until now considered to be an exclusive feature of blood vessel endothelium and hemopoietic cells, and 2) that this determinant appears in bursal epithelium at the time and site of hemopoietic cell invasion. The other point arising from this work concerns the apparent constitutive Ia expression by perifollicular blood capillary endothelial cells in normal and chimeric bursas.     

关于间质生血干细胞侵入法氏囊上皮及分化成淋巴细胞的研究

在上世纪末Retterer(1885)认为法氏囊的淋巴细胞是法氏囊上皮本身发生的。到本世纪初有人认为从法氏囊间质的“原始成血细胞”侵入上皮分化而成的(Jolly,1915)。直到六十年代初,Ackerman和Knouff(1959),Ackerman(1962),还认为法氏囊髓部的淋巴细胞是上皮细胞发生的,而皮部的淋巴细胞是法氏囊间质细胞及未分化的上皮发生的。用染色体标记等技术证明生血干细胞是法氏囊淋巴细胞的先躯细胞(Moore和Owm1965,1966;Jaffe和Fechhelmer,1966;Le Douarin和Houssaint,1974以及Houssaint等,1976)。生血干细胞在鸡胚发育三天到四天就存在于血液中,只有在法氏囊原基发育到一定阶段才开始侵入(Le Douarin等,1976)。在鸡胚从孵化8天到14天侵入法氏囊原基     

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.     

Modulation of glucocorticoid hormone receptor levels in chicken lymphoid tissue following treatment with androgens in vivo

Lymphatic tissues are highly sensitive to androgens and androgens are thought to contribute to sex differences in the immune response. In this study we have examined the effects of androgens on cytosolic glucocorticoid receptor levels in lymphoid tissues. The immature chick was chosen for our experimental model because it allows the separate evaluation of the bursa of Fabricius (primarily B-cells) compared to the thymus (primary T-cells). Treatment with dihydrotestosterone (a potent androgen in chicks) for 3–12 days in vivo reduced the cytosolic glucocorticoid (triamcinolone acetonide-[³H]) receptors in the bursa tissue to ∼ 42% of control levels after 5 days and ⩽ 5% of control levels after 7 days of treatment. The chick thymus tissues were still ~ 92% of control triamcinolone acetonide receptor levels after 5 days of androgen treatments. However, the thymus levels had dropped to ⩽ 5% of control values after 12 treatment days. Thus a difference in the rate of decrease in the bursa of Fabricius compared to the thymus was indicated. The blastogenesis index (BI), a measurement of the percentage of cells progressing through the cell cycle, was figured using fluorescent DNA staining with diamidino phenylindole followed by flow cytomctry analysis. After 3, 5, or 7 days of androgen treatment, the bursa of Fabricius from dihydrotestosterone treated chicks (2 mg/day/chick) had a mean BI = 11.17 (±3.07 SD) which was significantly lower than the bursa of Fabricius from control chicks which showed a mean BI = 27.33 (±3.42 SD). The thymus from dihydrotestosterone treated chicks had a mean BI = 19.57 (±2.19 SD) which was slightly but not significantly higher than the control thymus BI = 17.38 (±0.89 SD). In summary, androgen treatment in vivo induced a decrease in the cytosolic glucocorticoid hormone receptor levels in both the chick thymus and bursa of Fabricius tissues while decreasing the blastogenesis index in the bursa cells but not in the thymus cells.     

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.     

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.     

Perinatal deletion of B cells expressing surface Ig molecules that lack V(D)J-encoded determinants in the bursa of Fabricius is not due to intrafollicular competition

During embryonic development, the avian bursa of Fabricius selects B cell precursors that have undergone productive V(D)J recombination for expansion in oligoclonal follicles. During this expansion, Ig diversity is generated by gene conversion. We have used retroviral gene transfer in vivo to introduce surface Ig molecules that lack V(D)J-encoded determinants into B cell precursors. This truncated mu heavy chain supports both B cell expansion within embryo bursal lymphoid follicles and gene conversion. We show that individual follicles can be colonized exclusively by cells expressing the truncated mu chain and lacking endogenous surface IgM, ruling out a requirement for V(D)J-encoded determinants in the establishment of bursal lymphoid follicles. In striking contrast to their normal development in the embryo, bursal cells expressing the truncated mu-chain exhibit reduced rates of cell division and increased levels of apoptosis after hatching. The level of apoptosis in individual follicles reflects the proportion of cells within the follicle that express the truncated mu-chain. In particular, high levels of apoptosis are associated with follicles containing exclusively cells expressing the truncated micro receptor. Thus, apoptotic elimination of such cells is not due to competition within the follicle by cells expressing endogenous surface IgM receptors. This provides the first direct demonstration that the regulation of B cell development in the avian bursa after hatching differs fundamentally from that seen in the embryo. The requirement for intact IgM expression when the bursa is exposed to exogenous Ag implicates a role for Ag in avian B cell development 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.     

IgG Fc receptor-bearing cells during early lymphoid cell development in the chicken

Cells from intraembryonic mesenchyme, yolk sac, bursa of Fabricius, and thymus from chicken embryos at different stages of development were studied for the presence of IgG Fc receptors by EA-rosette formation and binding of heat-aggregated chicken IgG (agg IgG). Cells with Fc receptors were found in high frequency in the intraembryonic mesenchyme as early as on the third day of incubation, in the yolk sac on the 7th day, in the bursa on the 10th day, and in the thymus on the 16th day of embryonic development. In the bursa the number of agg IgG binding cells increased with the age of the embryo and remained high after hatching, whereas in the thymus the peak value (76%) was observed on the 16th embryonic day, and after hatching only about 10% of the cells expressed the agg IgG receptors. The results also suggest that the appearance of IgG Fc receptors precedes the expression of B-L (Ia-like) antigens and of cytoplasmic and surface immunoglobulins on early lymphoid cells of the chicken embryo.     

Heterotypic induction of stomach-like glands in the urogenital sinus endoderm under the influence of stomach mesenchyme of foetal rats

Summary Urogenital sinus endoderm of 16.5-day rat foetuses was combined with stomach mesenchyme and the recombinants were either treated with testosterone and grown in vitro or cultured beneath the kidney capsule of adult male rats of the same strain. It was found that testosterone stimulated mitosis in the urogenital endoderm. In recombinants grown under the kidney capsule a stratified squamous epithelium and stomach-like glands were induced under the influence of the forestomach and glandular stomach mesenchymes. However, the induced glands expressed neither rat pepsinogen nor rat ventral prostatic antigen. They did not produce mRNA for the prostatic steroid-binding protein C1. Thus, stomach mesenchyme of rat foetuses induces organ-specific morphogenesis but not functional differentiation in the heterologous endoderm, indicating that cytodifferentiation does not always accompany morphogenesis.     

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.     

Prebursal stem cells in the intraembryonic mesenchyme of the chick embryo at 7 days of incubation.

Cyclophosphamide-treated 18-day-old chick embryos were transplanted with cells from 7-day intraembryonic mesenchyme; the recipients and donors were identical at the major histocompatibility locus. At the age of 35 days, the cell recipients were studied to determine the reconstitution capacity of the transplanted cells. The transplantation resulted in a complete restoration of IgM and IgG class antibody production against human gammaglobulin and Brucella abortus, and of microscopic morphology of the bursa of Fabricius and of the germinal center formation in the spleen. These findings demonstrate that 7-day intraembryonic mesenchyme of the chick embryo harbor prebursal stem cells. These findings confirm our previous observations in the yolk sac-embryo chimeras indicating that lymphoid stem cells originate in the intraembryonic hematopoietic sites.     

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.     

Hormonal, cellular, and molecular regulation of normal and neoplastic prostatic development

This review on normal and neoplastic growth of the prostate emphasizes the importance of epithelial-mesenchymal/stromal interactions. Accordingly, during prostatic development urogenital sinus mesenchyme (a) specifies prostatic epithelial identity, (b) induces epithelial bud formation, (c) elicits prostatic bud growth and regulates ductal branching, (d) promotes differentiation of a secretory epithelium, and (e) specifies the types of secretory proteins expressed. In reciprocal fashion, prostatic epithelium induces smooth muscle differentiation in the mesenchyme. Epithelial-mesenchymal interactions during development continue postnatally into adulthood as stromal-epithelial interactions which play a homeostatic role and in so doing reciprocally maintain epithelial and stromal differentiation and growth-quiescence. Prostatic carcinogenesis involves perturbation of these reciprocal homeostatic cell-cell interactions. The central role of mesenchyme in prostatic epithelial development has been firmly established through analysis of tissue recombinants composed of androgen-receptor-positive wild-type mesenchyme and androgen-receptor-negative epithelium. These studies revealed that at the very least ductal morphogenesis, epithelial cytodifferentiation, epithelial apoptosis and epithelial proliferation are regulated by stromal and not epithelial androgen receptors. Likewise, progression from non-tumorigenesis to tumorigenesis elicited by testosterone plus estradiol proceeds via paracrine mechanisms. Thus, stromal-epithelial interactions play critical roles in the hormonal, cellular, and molecular regulation of normal and neoplastic prostatic development.