Cytophilic properties of surface immunoglobulin of thymus-derived lymphocytes

The cytophilic properties of released surface immunoglobulins of normal thymus lymphocytes and of activated thymus-derived lymphocytes (ATC) were analysed by lactoperoxidase-catalysed radioiodination in conjunction with immunological and autoradiographic techniques. Immunoglobulin from both normal T cells and ATC was cytophilic for macrophages (peritoneal exudate cells), but showed no detectable capacity to bind to either T lymphocytes or to bone-marrow-derived lymphocytes (B cells). Under the operative experimental conditions surface immunoglobulin of B cells did not show appreciable binding to macrophages. These results support the feasibility of models of collaboration between T cells and B cells which involve a soluble antigen-specific collaborative factor (T-cell Ig complexed with antigen) and show an obligatory requirement for macrophages.     

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.     

Antigen-binding thymus-derived lymphocytes: II. Nature of the immunoglobulin determinants

Thymus-derived `rosette'-forming lymphocytes which have been separated from other SRBC-sensitive cells by means of cotton wool columns were examined for the presence of immunoglobulin. This was carried out by inhibition of rosette formation by anti-immunoglobulin sera. Inhibition was effected by a number of anti-IgM sera shown to contain antibodies with specificities directed towards the `hinge' region of the μ chain. No other heavy chain specific antisera were inhibitory.

The ratio of rosette inhibition by anti-κ and anti-λ light chain sera varied during the course of the response to SRBC, the latter inhibiting by 89 per cent 3 days post-immunization.

     

Independent movement of surface immunoglobulin from Fc receptors on lymphocyte membranes.

The majority of surface immunoglobulin-positive lymph node cells possess Fc receptors detectable by a rosette technique. The movement of surface immunoglobulin to form caps does not alter the distribution of Fc receptors, although Fc rosette-forming indicator cells collect over the immunoglobulin cap under capping conditions.     

Detection of bursa and thymus-specific alloantigens in the chicken.

CH strain chickens selected from the F2 progeny of a cross between CH (B10/10) and B14B (B14/14) strains carried bursa (BA) and thymus (TA)-specific alloantigens of B14B parental origin. The BA marker was present on 95% of bursal cells but only on 10% of splenic and blood B lymphocytes. The TA marker was expressed by 80% of thymus cells and weakly by 45% of splenic and 70% of blood T lymphocytes. The adopted immunological and genetic protocol offers a feasible approach towards detection of differentiation antigens in major histocompatibility complex-homozygous but 'minor' alloantigen-segregating populations.     

Morphological and functional differentiation of the surface epithelium of the bursa Fabricii in chicken

Morphological and functional differentiation of the mucosal surface epithelium of the bursa Fabricii was studied in White Leghorn chicken fetuses and newly hatched chickens. First signs of differentiation towards two types of epithelial cells appeared on the thirteenth day of incubation: The apical cells of the epithelial buds projected towards the lumen, and an increase in the number of Golgi regions was observed in the epithelial cells between the buds. On day 15 the follicle-associated epithelium contained small apically situated vacuoles, and large mucin granules appeared in the interfollicular surface epithelium. Towards the day of hatching both epithelial cell types were arranged to a monolayered or pseudostratified cylindrical epithelium. The follicle-associated epithelium had invaginations and small vacuoles in the apical cytoplasm, whereas the interfollicular surface epithelium had numerous microvilli on its apical surface and large mucin granules in the apical cytoplasm. In functional studies, endocytosis of colloidal carbon was demonstrated in four out of ten 19-day-old fetuses and in all chickens studied immediately after hatching.     

Comparison of lymphocyte populations bearing surface immunoglobulins in avian bone marrow, bursa, spleen and thymus.

Rabbit anti-chicken gamma-globulin was labeled with 125I and then incubated with cells from the bursa, thymus, spleen, and bone marrow of 4- and 8-week old birds. The same procedure was carried out on 11-week-old agammaglobulinemic chickens. Autoradiography revealed that the majority of large, medium, and small bursal lymphocytes bind the antibodies while labeled lymphocytes of each type in the spleen and thymus never exceeded 11 or 4 percent, respectively. Labeled medium and small lymphocytes in the bone marrow increased from 4.2 and 1.7%, respectively, at 4 weeks of age, to 9.5 and 8.8%, respectively, at 8 weeks of age. Labeled lymphocytes of all sizes were completely absent in all tissues of agammaglobulinemic chicks, including the marrow. Therefore, the increase in frequency of labeled lymphocytes in the bone marrow with age may be a result of recruitment of cells from the bursa of Fabricius. The majority of lymphocytes in the bone marrow do not label. Therefore, lymphocytes from the bone marrow may be T cells, subsets of B cells, or neither T or B cells.     

Induction of B lymphocyte colony growth in vitro by thymus-derived stimulating factor.

Murine lymphoid cells which were stimulated in liquid culture containing thymus culture fluid (Thy-CF) and seeded in a soft agar culture system, proliferated and developed into B cell colonies. Two types of colonies were formed: large colonies within the upper layer and small flat colonies on the surface of the upper layer. Thy-CF prepared from cells of normal hydrocortisone-treated mice had a higher cloning potential than Thy-CF prepared from untreated mice. At concentrations of Thy-CF in culture medium greater than 35%, Thy-CF prepared from normal mice had an inhibitory effect on colony formation. Cells of nude mice were also able to form B cell colonies if thymocytes of normal mice were mixed with lymphoid cells in the culture medium. Thymocytes elaborate a B lymphocyte colony-stimulating factor which, with the help of T cells, triggers a B cell population into colony formation and immunoglobulin production.     

Purine metabolism and B-lymphocyte development in the chicken bursa of fabricius.

The activity of three enzymes involved in the salvage pathway of purine nucleosides--purine nucleoside phosphorylase (PNP), xanthine dehydrogenase (XDH), and hypoxanthine-guanine phosphoribosyl transferase (HGPRT)--was investigated in cellular fractions of the chicken bursa of Fabricius differentially enriched in epithelial cells or lymphocytes. Markedly increasing levels of PNP and XDH were observed along with the enrichment in epithelial cells together with a slight, though significant, decrease in HGPRT activity. By contrast, a dramatic fall in PNP and XDH activities was detected along with the enrichment in lymphocytes together with a slight, though significant, increase in HGPRT activity. This sharply different distribution of the three enzymes, all sharing hypoxanthine as a substrate, clearly indicates that lymphocytes preferentially channel hypoxanthine into the salvage and interconversion pathways, phosphorylating it to IMP, while epithelial cells rapidly catabolize such a purine base to uric acid. Moreover, epithelial cells, unlike lymphocytes, are able to retain high intracellular levels of both hypoxanthine and inosine. These results support the possibility that epithelial cells contribute to the normal development of bursal lymphocytes by supplying such actively proliferating cells with purine rings and at the same time by preventing them from accumulating potentially toxic high levels of purine nucleotides being able to rapidly eliminate excess hypoxanthine as uric acid from the bursa environment into the bloodstream.     

Emigration of B cells from chicken bursa of Fabricius

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.     

Cross-linking of the surface immunoglobulin on lymphocytes from the bursa of Fabricius results in second messenger generation

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.     

The binding of LPS to the lymphocyte surface.

Bacterial lipopolysaccharide (LPS) from Escherichia coli labelled with tritium has been used to follow the binding of LPS to lymphocytes. Binding to cells rose to a maximum 2-7 min after addition of [3H]-LPS, followed by loss of [3H]-LPS from cells, reducing to about 10% of the peak level at 20-30 min. Peripheral blood lymphocytes, mesenteric lymph node and thymus cells of the pig and CBA, C3H/He and C3H/HeJ mouse spleen cells all bound [3H]-LPS transiently at similar levels. It is concluded that this type of LPS binding cannot be solely responsible for the preferential stimulation of B cells by LPS.     

Proteolysis of lymphocytic surface immunoglobulin.

Limited proteolysis of lymphocytic surface immunoglobulins in guinea-pig, rabbit and man was investigated by immunofluorescence using conjugated antisera specific for immunoglobulin fragments. The cell surface IgM of guinea pig L2C leukaemic lymphocytes and rabbit blood lymphocytes was cleaved in situ at its hinge region by papain. The Fcmicron fragment remained attached to the membrane and could be stained with the appropriate anti-Fc conjugate. The surface IgD and IgM of human chronic lymphocytic leukaemia cells was cleared from the cell surface by papain, as shown by reagents directed against both Fab and Fc region determinants. This could be due either to proteolytic degradation of membrane bound Fc or to initial cleavage of Ig from the membrane at some point other than the hinge region.     

Cytophotometric characterization of cortical and medullary lymphocytes in the chicken bursa and thymus.

We have evaluated by means of cytophotometric techniques the nuclear content in DNA, in total nucleic acids and in histone proteins and the nuclear volume of the thymic and bursal lymphocytes in adult chickens. We have also made the same determinations in the developing bursal follicles before and after hatching. The results indicate that cortical differ from medullary lymphocytes for all the parameters considered both in thymic lobules and bursal follicles. Furthermore these differences appear analogous in both the organs, independently from the fact that they produce precursors of T and B lymphocytes respectively. As concerns the developing bursal follicle, the lymphocytes show the characteristics of the adult medullary lymphocytes. At the hatching changes occur in the nuclear content of total nucleic acids and histones. This is probably related to the exposure to antigenic stimulation through the cloaca.     

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.