Class switch recombination of the chicken IgH chain genes: implications for the primordial switch region repeats.

In mammals and the amphibian, Xenopus, isotypes of antibodies have been shown to be changed through class switch recombination within the IgH chain gene locus. Here, we identified switch (S) repetitive sequences in the 5' introns of the Ig C(mu) and C(gamma) genes of the chicken. The S(mu) region is composed of two homologous regions, S(mu)1 and S(mu)2. The S(mu)1 region is an upstream 3.7 kb sequence composed of 37 repeats of a consensus sequence containing tandem repeats of the decamer ACCAGTATGG. The S(mu)2 region is a downstream 1.4 kb sequence consisting of simple tandem repeats of a decamer CCCAGTACAG. The S(gamma) region contains repeats of the decamer TATGGGGCAG. Analysis of chicken IgG-producing hybridomas revealed that the C(mu) gene was deleted from the chromosome by the recombination occurring between the S(mu) and S(gamma) regions. Recombination breakpoints at the C(mu) gene of splenocytes from an immunized chicken were scattered around the S(mu) region and two such breakpoints, the precise position of which were determined, were located within possible hairpin loop structures at the palindromic sequence of S(mu)1. A primordial palindromic sequence from which the prevalent switch repeat motifs of mammals, chickens and amphibians may have diverged is presented.

A comparative study of germinal center: fowls and mammals.

The mammalian germinal center is organized into a dark zone containing proliferating centroblasts and a light zone filled with nondividing B cells (called centrocytes), follicular dendritic cells and a few scattered T cells. We clarified these two zones in the chicken germinal center with immunohistology. Proliferating cells and immunoglobulin negative cells were detected in the circumference ring of the chicken germinal center. The central part of the chicken germinal center contained B cells expressing immunoglobulin, follicular dendritic cells and a few T cells. Most of the B cells in the central part of the chicken germinal center did not enter into the S phase. These results suggest that the chicken germinal center is also organized into the dark zone (the circumference ring of germinal center) and the light zone (the central part of germinal center).

Development of maternal IgG-free chick obtained from surgically bursectomized hen.

IgG-free eggs and chicks were developed, so as to study the role of maternal IgG in the development of the immune system. Surgical bursectomy on the 18th day of incubation deprived chickens of B cells and eliminated IgG synthesis. Bursectomized chickens are usually dead before sexual maturity under conventional conditions. When surgically bursectomized chickens were housed in an isolated clean room and antibiotics were administered to them, they could survive to sexual maturity. Finally, we succeeded in obtaining IgG-free fertilized eggs and maternal IgG-free chicks from surgically bursectomized hens. The amount of yolk IgG in IgG-free eggs was one-ten thousandth less than that in normal eggs. The level of IgM in the serum of maternal IgG-free chicks reached six times higher than that of normal chicks 5 days after hatching.

Oligoclonal development of B cells bearing discrete Ig chains in chicken single germinal centers.

Chicken single germinal centers enable us to analyze the postbursal diversifications of B cells due to their easy isolation. Germinal center formation has peaked by day 7 of primary responses and begins to wane 14 days after immunization. To detail the kinetics of Ig mutation and selection, we analyzed Ig light chain sequences recovered from single germinal centers at 7 and 11 days postimmunization with an artificial Ag. Our observations show that multiple, Ag-activated B cells migrating into single germinal centers are diversified by gene conversion in the very early phase of the germinal center reaction and are subsequently subjected to point mutations and selection for oligoclonality.

Immunoglobulin gene hyperconversion ongoing in chicken splenic germinal centers.

It has been believed that the peripheral lymphocytes in chickens proliferate by self-renewing amplification of the preimmune repertoire generated in bursa. We amplified rearranged immunoglobulin variable (V) region genes from the single germinal centers induced by immunization. The sequence analysis of these genes revealed that most were derived from distinct B-cell clones which expanded locally, generating somatic antibody mutants at a high rate. Somatic hypermutations included unlinked base changes and the linked base modifications interpreted as unidirectional transfer of sequences from V region pseudogenes. This finding demonstrates the ongoing post-bursal diversification of B-cells in splenic germinal centers by templated gene conversion as well as untemplated point mutations.

Role of environmental antigen in the development of IgG+ cells in the bursa of fabricius.

IgG+ cells were detected in the bursa of Fabricius after hatching by immunofluorescence staining of single cells and immunohistologic studies using mAb anti-Ig gamma-heavy chain. The frequency of IgG+ cells in the bursa increased rapidly immediately after hatching. In histologic studies, most of the IgG+ cells were found in the medullary areas of the bursal follicle. Isolation of the bursa from the gut by bursal duct ligation before hatching suppressed the development of IgG+ cells in the bursa after hatching. In addition, administration of Ags into the bursal lumen just before bursal duct ligation caused a significant increase of IgG+ cells in the bursa compared with the isolated bursa. However, parenteral administration of Ags had no effect on the frequency of IgG+ cells in the isolated and normal bursa. These results suggest that Ags in the bursa are a prerequisite for the development of IgG+ cells in the bursa. Although the majority of IgG+ bursal cells were IgM+ IgG+ double-positive cells, sorted Bu1+ bursal cells, which contained 99.9% of IgG+ cells but not plasma cells, synthesized de novo IgM but little or no IgG. Therefore, it is likely that surface IgG on the bursal cells is not produced locally, but is trapped by IgM+ bursal cells. We speculate that Ags derived from the bursal lumen form immune complexes within the bursa, which are subsequently trapped on the surface of IgM+ bursal cells mediated either by Fc-receptors, C3 receptors, or surface Ig.

Expansion of methylmercury poisoning outside of Minamata: an epidemiological study on chronic methylmercury poisoning outside of Minamata.

The first methylmercury poisoning by consumption of fish arose in Minamata, Japan, in 1953. Methylmercury dispersed from Minamata to the Shiranui Sea until 1968. Mercury concentration in the hair of residents on the coast of the Shiranui Sea was 10 to 20 times higher than that in nonpolluted people in Kumamoto Prefecture in 1960. People on the coast of the Shiranui Sea have consumed fish containing low-dose methylmercury without a ban over decades until 1968. We studied the effect of long-term consumption of methylmercury on those people 10 years later after the end of methylmercury dispersion. Our epidemiological study clarified that people in a fishing village (Ooura) on the coast of the Shiranui Sea showed a significantly higher frequency of neurological signs characteristic of methylmercury poisoning (hypoesthesia, ataxia, impairment of hearing, visual change, and dysarthria) in comparison with people in a nonpolluted fishing village (Ichiburi). The neurological disorders were still detected 10 years later in Ooura after the end of methylmercury dispersion from Minamata; hypoesthesia showed the highest frequency in Ooura. These results suggest that people on the coast of the Shiranui Sea were afected by long-term dietary exposure to methylmercury.

Role of environmental antigens in B cell proliferation in the bursa of Fabricius at neonatal stage.

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.

Monoclonal antibodies as probes for defining cellular subsets in the bone marrow, thymus, bursa of fabricius, and spleen of the chicken.

Using immunohistochemistry, the distribution and characteristics of cells detected by the newly developed monoclonals HIS-CI (B lymphocytes), HIS-C7 (leucocytes), HIS-C12 (IgM), CVI-ChIgM-59.7 (IgM), CVI-ChIgG-47.3 (IgG), and CVI-ChIgA-46.5 (IgA) are described in bone marrow, thymus, bursa of Fabricius, and spleen of chickens of different ages. Furthermore, quantification of cells positive with the described monoclonal antibodies was performed on cytocentrifuge preparations. The specificities of the monoclonal antibodies are discussed.

The bursa of Fabricius: a trapping site for environmental antigens.

The entry of environmental antigens into the lumen of the bursa of Fabricius was prevented by ligating the bursal duct prior to hatching (BDL: bursal duct ligation). The development of 'natural' serum agglutinins for bacteria and heteroerythrocytes was markedly suppressed by BDL. The antigen-specific recovery was observed by simultaneous administration of sterile antigens into the bursal lumen at the time of BDL. These results strongly suggest that the bursa of Fabricius is a major channel through which environmental antigens stimulate the immune system and induce the formation of 'natural' serum agglutinins.

Migration of macrophages from the marginal zone to germinal centers in the spleen of mice.

Histological observations of the mouse spleen were carried out at different times after intravenous carbon injection. Large carbon-laden macrophages appeared in great numbers in the marginal zone soon after injection. They came together favorably around the germinal centers. Possible migration of these cells toward the germinal centers diffusely from the periphery of the white pulp or through the periarterial lymphoid sheath was suggested. These macrophages entered the germinal centers on a large scale and clustered for a long period--at least 180 days. Since the same type of macrophages were observed persistently in the marginal zone, it was thought that some of them might arise from the blood stream. Possible migration of these cells from the marginal zone toward the germinal centers was also persistently observed. A second type of much smaller carbon-laden macrophages was seen in the white pulp. However, they never showed any favorable localization in the germinal centers as did large carbon-laden macrophages.

The bursa of Fabricius as a trapping mechanism for environmental antigens.

The bursa of Fabricius was isolated from gut-derived antigens by ligating the bursal duct on the 18th day of incubation. Ligation of the bursal duct (BDL) suppressed the bursal development, spontaneous germinal centre formation in the spleen and the development of serum "natural" agglutinins for bacteria or heteroerythrocytes. Moreover, administration of sterilized cecal contents into the bursal lumen at BDL led to normal level of serum "natural" agglutinins. These findings strongly suggest that the bursa of Fabricius possesses an antigen-dependent process which modulates the development of immune system. Antigen-trapping mechanism of the bursa may supply information about environmental antigens for this process.

Suppression of immune response by isolation of the bursa of Fabricius from environmental stimuli.

When the bursa of Fabricius was isolated from environmental stimuli across the bursal epithelium by clipping the bursal duct on the 19th day of incubation, marked retardation was observed both in the bursal development and in the maturation of the immune reactivity. Neither necrotic changes nor involution in the bursa was, however, observed at any stage examined after clipping the bursal duct. Lymphopoiesis in the bursa and the immune reactivity of these chickens gradually increased with age, although they were constantly lower than those of controls. These results suggest that environmental stimuli across the bursal epithelium play an important role in teh amplification mechanisms of B-cell differentiation in the bursa.

Distribution and role of lymph vessels of the bursa Fabricii.

The fine distribution of lymph vessels in the bursa of Fabricius and the efferent lymph vessels coming from the bursa were demonstrated. The lymph vessels never entered lymphoid follicles, whereas the blood vessels pierced the cortex of lymphoid follicles and formed a network at the boundary between the cortex and medulla of lymphoid follicles. The lymph vessels commenced in the immediate exterior of lymphoid follicles. A large number of lymphoid cells were found in the lumen of the lymph vessels. Migration of lymphoid cells from the cortex of lymphoid follicles to the lymph vessels was also observed. Therefore, it was assumed that the lymph vessels of the bursa are important in the transport of lymphoid cells from the bursa to the blood stream.

Sex differences in the formation of germinal centres in the rat thymus.

Rats of inbred DA strain were given 20 intraperitoneal injections of typhoid-paratyphoid bacilli at 10 day intervals. Thymic lymph follicles with germinal centres were formed in females but not in males. However, orchidectomy permitted the formation of large numbers of thymic lymph follicles with germinal centres in males. The formation of lymph follicles with germinal centres in the thymus was not affected by ovariectomy. These results would appear to suggest an inhibitory role of androgens on the formation of thymic lymph follicles with germinal centres. In addition, large numbers of plasma cells, mast cells, and reticular cells, or macrophages with haemosiderin in their cytoplasm, appeared in the parenchma of the thymus in close association with the formation of thymic germinal centres.

Amplification of plaque-forming cells in the spleen after intracloacal antigen stimulation in neonatal chicken.

Neonatal chickens were primed by multiple or a single injection with sheep red blood cells (SRBC) into the cloacal lumen and then were challenged by intravenous injection with the same antigen. Direct plaque-forming cells (PFC) in the spleen were markedly increased shortly after intracloacal priming. This enhancing effect was antigen specific and was abolished by surgical bursectomy 24 h after intracloacal priming. A similar effect was also found in mature adult chickens, although the degree of enhancement was far less than that found in neonatal chickens. These results suggest that a certain stage of B-cell differentiation in the bursa of Fabricius (BF) is regulated by antigenic stimulation.