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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.

Effect of environment antigens on S phase entry rate of bursa cells was studied by utilizing in vivo incorporation of bromodeoxyuridine (BrdUrd). S phase entry rate of bursa cells in chick embryo was determined to be 6.4%/h, this rate increased to 10.0%/h at neonatal stage and decreased to 6.4%/h 6 weeks after hatching. Evidence is presented that isolation of the bursa from the gut before hatching suppressed S phase entry rate of bursa cells after hatching, whereas S phase entry rates of thymocytes were not affected. On tissue sections, a decrease of BrdUrd+ cells was observed in medullary areas of the isolated bursa. It is proposed that in situ stimulation of bursa cells by environmental antigens to the bursa accelerates S phase entry rates, and that medullary areas are the major sites for antigen-driven B cell proliferation after hatching.

In chickens, the pre-B cell development takes place in a specialized organ, the bursa of Fabricius. In this study, we show that fibronectin can be detected in the bursa at least from the day 15 of embryonic incubation up to 10 weeks of age after which the bursa starts to involute. During the embryonic incubation, fibronectin localizes mainly in the areas between the follicles and later on also in the border zone between the cortical and medullary parts of the lymphoid follicles. By adhesion studies with synthetic peptides we show that immature bursal B cells bind in a dose-dependent manner to the CS-1 site of the type III connecting segment at the carboxy-terminal end of fibronectin throughout their bursal development. Postbursal B cells from chicken spleen also display a similar preference of binding to the CS-1 site of fibronectin, whereas peripheral blood B lymphocytes bind to the 40-kDa fragment of fibronectin, but do not adhere as strongly to the CS-1 site. Instead, they recognize to some extent also the heparin-binding YEKPGSPPREVVPRPRPGV peptide. The adhesion of the bursal B cells to the CS-1 site can partially be blocked by preincubation of cells with a synthetic CS-1 peptide and also by preincubation with a monoclonal anti-fibronectin receptor antibody. In contrast, the RGD motif present in several adhesion-associated stromal molecules did not promote any adherence of bursal lymphocytes. The bursal B cells which adhere to the fibronectin molecule in vitro form only a small fraction of the total number of B cells present in the bursa as only less than 4% of the cells remained bound to the surface coated with fibronectin in the adhesion studies. In conclusion, these results suggest that fibronectin may be involved in the differentiation of pre-B cells in the bursa of Fabricius and also in the adherence of postbursal B cells in the spleen.

The ileal Peyer's patch (PP) and the bursa of Fabricius have major roles in populating the B cell system in sheep and chickens, respectively. These tissues contain greater than 90% B cells and possess a massive proliferation index with greater than 5% of B cells entering mitosis per hour. Paradoxically, almost all of the B cells produced in these sites rapidly die in situ. Here we show that the extensive B cell death occurring in the ileal PP and bursa is associated with apoptosis. Gel electrophoresis of ileal PP cell DNA from 7-14 week-old lambs and bursal cell DNA from 4-week-old chickens demonstrated a laddering of DNA in multiples of approximately 200 bp, a pattern indicative of apoptosis. In sheep, the intensity of the laddering pattern seen after agarose gel electrophoresis was always greater with ileal PP cell DNA compared with thymocyte DNA, and usually greater than jejunal PP cell DNA. Likewise, DNA isolated from chicken bursal cells and mouse PP cells always exhibited a more intense laddering pattern than chicken or mouse thymocytes, respectively. When placed in culture ileal PP cells died rapidly less than 40% viable cells were recovered after 24 h. Within 6 h of culture many ileal PP cells exhibited an apoptotic appearance in that they contained condensed chromatin and fragmented nuclei. Moreover, greater than 55% of total cellular DNA was fragmented. Compared with thymocytes, ileal PP cells underwent DNA fragmentation to a much greater extent and with a faster time course in short-term culture. We propose that cell death by apoptosis may make an important contribution to B cell development in the lamb ileal PP and the chicken bursa. Apoptosis may provide a mechanism for the diversification of the B cell immune repertoire and/or the selection of non-self reactive B cells.

The bursa of Fabricius represents the major site of B lymphocyte development in avian species. Although the vast majority of bursal lymphocytes express cell surface immunoglobulin (sIg), it is generally considered that the bursa does not represent a significant site of antigen-induced B cell maturation to Ig secretion. However, the question as to whether antigen, either exogenous or self, can induce positive or negative selection of bursal lymphocytes in such a way as to modify the peripheral B cell repertoire remains open. Clearly, such intrabursal selection would require that bursal lymphocyte sIg have the molecular machinery to transduce signals into the cell as a consequence of its interaction with antigen. In this report we demonstrate that exposure of bursal lymphocytes to antibodies directed against sIg induced a rapid increase in cytosolic free calcium ion concentrations [Ca2+]i. Furthermore, such antibodies also induced a rapid increase in intracellular phosphatidic acid concentrations followed by a rise in intracellular phosphatidylinositol. Increased [Ca2+]i, phosphatidic acid and phosphatidylinositol levels required the cross-linking of sIg and were not induced by antibodies to other bursal cell surface antigens. Thus, cross-linking of the sIg on bursal lymphocytes results in second messenger generation, demonstrating that bursal sIg is a functional signal transduction element.

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.

Treatment of neonatal chickens with cyclophosphamide depletes bursal lymphocytes while maintaining the bursal epithelium intact. The bursae of normal young chickens contain "bursal stem cells" which can reconstitute the lymphoid compartment in the bursa of the cyclophosphamide-treated recipient. Using bursal stem cells from IgM allotype-heterozygous donors we show that most bursal follicles in the reconstituted host are colonized by single stem cells which are committed to the expression of one or other IgM allotype. In addition we show that the reconstituting bursal stem cells express allelically excluded surface IgM at the time of transfer. Our results suggest that B lymphocyte numbers in hatched chickens are maintained by self-renewal of committed precursors rather than by de novo production from multipotential stem cells.

The metaphase arrest technique was used to quantitate the mitotic rate in sheep Peyer's patches (PP) and in the chicken bursa of Fabricius. It was shown that the mitotic rate in these tissues was the same whether or not the animal had been exposed to extrinsic antigen. The mitotic rate was calculated from the rate of accumulation of metaphase cells in tissue sections taken up to 4 h after i.v. vincristine. The follicular B cells in PP of fetal sheep at 3-5 days before birth, and in the bursas of chick embryos 2-3 days before hatching, none of which had experienced extrinsic antigen, entered mitosis at a rate of 5.2%/h and 7.5%/h, respectively. A similar analysis was done on animals at approximately 6 weeks of age, at which stage the PP and bursa had received chronic exposure to antigen from the gut. The mitotic rates were not significantly different from those in fetuses, i.e., 5.4%/h for PP and 6.0%/h for the bursa. In the follicles of both young lambs and juvenile chickens, the mitotic rate was 2-3 times greater in the outer, or cortical zone, than in the inner, or medullary zone. In a similar study of the thymus, no significant differences were found between the mitotic rates in fetal sheep and young lambs. The mitotic rate for the thymus was 15-20-fold less than for the PP. It is proposed that sheep PP, the chicken bursa and the thymus have in common the important characteristic of establishing a maximal rate of lymphocyte division prior to being exposed to antigen.

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.

Previous studies have shown that the same immunoglobulin (Ig) V lambda gene (V lambda 1) is rearranged in all chicken B cells, and that extensive sequence diversification of this gene occurs during B cell development in the bursa of Fabricius. We used two-dimensional gel electrophoresis to compare the heterogeneity of Ig lambda light chains produced by B cells at different stages of bursal development. Somatically diversified light chains were observed in Ig molecules produced by bursal cells as early as 15 days of embryonic incubation. The two principal species of light chain observed probably represent glycosylated and nonglycosylated forms of lambda chain encoded by alleles of a single lambda gene. Extensive diversification was observed during late embryogenesis. We also studied lambda light chain diversity in cyclophosphamide-treated birds repopulated with normal bursal cells. In these birds, individual bursal follicles are repopulated by single B cell precursors. Follicular cells derived from single B cell precursors were able to produce a spectrum of light chains almost as diverse as that of the total bursal cell population. We used two monoclonal anti-idiotype antibodies to study idiotype expression in individual normal or reconstituted follicles. About 30% of follicles contained 0.1% to 5% of lymphocytes which reacted with one or both of the antibodies. The results indicate that within individual bursal follicles bursa stem cells undergo Ig hyperdiversification.

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.

A technique of surgical removal of the bursal primordium ("bursectomy") of chicken embryos at stage 17, approximately 52-64 hours and 29-32 somites, is described. The survival rate of bursectomized (Bx) embryos approached a level of 50% on the 21st day. About 20% of correctly Bx embryos exhibited malformations of the anal sphincter and the large intestine. Using a rabbit anti-bursacyte serum, which did not react with thymocytes, the specific bursa-derived cell (Bu) marker was detected on the surface of bursa, spleen, bone marrow and thymus lymphocytes. Early embryonic bursectomy caused a moderate depletion of Bu marker-bearing and IgM-containing cells. It has been postulated that embryonic Bu cells can be recruited from sites other than the bursa and in the absence of the bursa.

Chicken spleen and bursa cells were examined for the percentage of Fc receptor-bearing cells. Rosette formation was done with chicken 7S antibody-sensitized sheep erythrocytes and was inhibited by heat-aggregated chicken Ig. In the spleen, the percentage was found to increase with age to approximately 26% at 7 to 12 weeks. In contrast, only 3 to 5% of bursa cellss at this age demonstrated Fc receptors. Sleens from bursectomized chickens had 7--10% Fc receptor-bearing cells. In an attempt to determine a possible role of the C3 receptor on migration patterns, the effect of cobra venom factor (CVF) on the localization of transferred lymphoid cells was examined. Pretreatment of recipients with enough CVF to lower mean C3 levels to 11% of controls failed to affect follicular B cell localization in mice at either 24 or 48 h after transfer. Localization of thymus or bursa cells in chickens was similarly unaffected by CVF pretreatment. The possible roles of Fc and C3 receptors on migration of B lymphocytes into follicles and germinal centers were discussed.

Lymphocyte precursors in bursa of Fabricius, thymus and bone marrow (BM) of chick embryos were studied at different stages of incubation over 12-21 days, and for their state of commitment to B or T cell lines of development. Cell suspensions were fractionated on albumin gradients to remove nonlymphoid cells and incubated in vitro with bursopoietin, a specific inducer of B cells, or crude chicken thymus extract, a specific inducer of T cells, or ubiquitin, a nonspecific inducer. Precursors were identified by increases in numbers of cells bearing surface alloantigens as determined by immunofluorescence, either Bu-1 (specific to B cells) or Th-1 (specific to T cells). Precursors inducible to Bu-1+ cells were found in bursal cells and BM cells from all age groups but not in thymic cells. Precursors inducible to Th-1+ cells were found in thymic preparations and BM cells at all ages, but in significant numbers in bursa on day 12 only. Because B and T precursors were never found together in bursa or thymus, or only in very unequal amounts, it was concluded that precursors in these organs were not multipotential but were separately committed to one or other line of development. This argument did not apply to BM cells, for which other evidence was obtained. Bu-1+ cells were specifically induced in BM cells with bursopoietin and then removed by complement-dependent cytolysis wih anti-Bu-1 antiserum. When the remaining cells were incubated with ubiquitin, only Th-1+ cells were induced, showing that Bu-1 and Th-1 precursors were separately committed. Surface IgM was never induced on either bursal or BM lymphocytes. The Ia (or B-L) antigen was inducible on 12- to 21-day bursal cells, but could not be generated on BM cells until day 14 onwards. The pattern of occurrence of committed lymphocyte precursors in the developing chick embryo suggests that these cells are released into the circulation from both central lymphoid organs at their respective times of high lymphopoietic activity, and accumulate in the BM at least up to the time of hatching. Moreover, the presence of committed B precursors in bursa and committed T precursors in thymus at times and in quantities appropriate to the known features of avian lymphopoiesis leads us to conclude that in vitro induction is analogous to a true stage of in vivo B and T cell differentiation.

The extent of emigration of cells from the bursa of Fabricius to the periphery was estimated. Per anum application of fluorescein isothiocyanate to label bursal cells in situ was used. Migrant cells can be visualized on frozen sections or cell suspensions of peripheral organs by their fluorescence. The data show that at 2-3 weeks after hatching about 5% of bursal cells leave the bursa per day. Since the bursal cells divide rapidly, this indicates that the vast majority (95%) of bursal cells die in situ. Cells that leave the bursa are surface IgM positive and go first to peripheral blood and later into B cell areas of spleen, thymus and cecal tonsils. The results are also discussed on the basis of their implication for the generation of antibody diversity in the chicken bursa.

To examine the antibody-forming capacity of neonatally cyclophosphamide-treated chickens reconstituted with limiting numbers of B cell precursors, we analyzed their antibody responses to six unrelated antigens. Our results demonstrate that about 10 x 10(6) bursal cells are needed in this adoptive cell transfer model to restore normal immune competence to B cell-deficient birds. From our earlier data we know that with low repopulating cell numbers (less than 10 x 10(6) cells) developing bursal follicles are of clonal origin. Ten million cells repopulate about 40%, i.e. about 4 x 10(3) of the bursal lymphoid follicles. Assuming the clonal origin of the follicles these results imply that the B cell system of birds receiving this dose is derived from less than 5 x 10(3) precursor cells. At the lowest reconstituting dose (1.25 x 10(6) cells) most birds do not respond to the antigens studied. However, their B cell system is derived from only about 500 precursor cells. Because the antibody repertoire of a normal chicken was estimated to be at least 10(6) our results suggest that each precursor gives rise to a large number (greater than 200) of immunoglobulin V-region gene variants during its clonal proliferation in the bursa. Our results are thus consistent with the proposed "hyperconversion" mechanism of generation of antibody diversity in the chicken and provide quantitative data useful for estimating what such somatic modification rates might be.

An in vitro culture system for embryonic chicken bursa is described, in which development, as measured by follicle formation (site of B lymphocyte development), position of large basophilic cells (stem cells), and presence of lymphocytes and cells containing cytoplasmic immunoglobulin (B cells), was comparable to that of the bursa in vivo. Using this system, preliminary studies on B lymphocyte differentiation were made. These support the view that bursal lymphocytes develop from precursors that migrate through the surrounding mesenchyme and into the follicles, but indicate that at least some of these cells may be "pre-B cells" rather than pluripotent stem cells.

The activities of 8 S and 6 S DNA ligases have been studied in the chicken lymphoid cells of blood, spleen and bursa of Fabricius at different stages of development, from late embryonic life to about 3 months after hatching. These cells have been sorted with the fluorescence-activated cell sorter FACS II on the basis of size and T or B antigenicity (immunofluorescence). The light 6 S DNA ligase has been previously demonstrated to be associated to a late stage of differentiation of thymocytes. In the bursa, a unique form of 8 S DNA ligase is found during the whole period of observation. This form of enzyme remains in the B cells of the spleen until 3 weeks after hatching, but is never present in the blood B cells. As far as T cells are concerned, the light DNA ligase is present in the blood from 18-day embryonic life on. In the spleen T cells, on the contrary, this enzyme appears only 3 weeks after hatching. Before this stage, splenic T cells are devoid of any form of DNA ligase activity. These findings show biochemical differences in T and B lymphocytes colonizing the periphery, blood and spleen, and suggest, at least for the T cells at early stages, a heterogeneity in the degree of differentiation.

We have analyzed the embryonic development of a population of lymphoid cells that express a CD3 antigenic determinant in the cytoplasm but not on the cell surface. Since these cells lack T cell receptor (TcR) molecules, we have provisionally named them TCRO cells. Their development, expansion and distribution was investigated following transplantation of splenic and bursal fragments from chicken embryos into quail embryos. Since quail cells are not recognized by our panel of monoclonal antibodies against chicken TcR1, TcR2, TcR3, CD3, CD4 and CD8 molecules, these antibodies provided reliable markers for donor chick lymphocytes in the tissues of the quail recipients. Transplanted spleen and bursa both generated CD3+ cells, the number of which increased as a function of age. Notably, approximately half of these CD3+ cells expressed surface CD8, but none acquired TcR1 (gamma/delta), TcR2 (alpha/beta) or TcR3 expression. Since TCRO cells normally appear first in the spleen of 8-day chick embryos (E8), their generation in E6 splenic transplants indicated an extrathymic origin. The TCRO cells of chick splenic origin migrated to the spleen, bursa and thymus of the quail recipients. In six of seven chimeras acquiring CT3+ cells in the recipient thymus, these cells were restricted to the medulla and displayed the typical TCRO phenotype: CD3+CD8+TcR1-TcR2-TcR3-. These intrathymic TCRO cells also lacked the CT1 thymocyte antigen. We conclude that the TCRO cells represent a thymus-independent lineage of lymphoid cells that can migrate into a receptive thymus by rarely, if ever, differentiate into conventional T cells.