Thymic epithelium tolerizes chickens to embryonic graft of quail bursa of Fabricius

We demonstrated previously that isotopic and isochronic grafts of the quail bursa of Fabricius rudiment performed at 5 days of incubation (E5) into chick embryos resulted in the development of a chimeric bursa whose chick host B lymphocytes and accessory cells differentiated in a foreign, quail epithelial environment. Such animals reject their grafted bursa by the age of 2-3 weeks post-hatching (1,2). Isotopic embryonic grafts of the thymus epitheliomesenchymal anlagen from the quail donor of the bursal rudiment were carried out at E4.5 (before their colonization by hemopoietic precursor cells), following partial or complete host thymectomy. The quail thymic epithelial stroma was accepted and invaded by chick hemopoietic precursor cells that further differentiated into lymphocytes and dendritic cells. Tolerance of the foreign bursa was induced in such thymobursal chimeras. This demonstrates that the thymic epithelium has the capacity to induce tolerance of xenogeneic rudiments when both grafts are implanted at early stages of embryonic development. We also report on the production of two birds in which removal of the chick host thymus was complete thus generating chimeras in which host T and B lymphocytes differentiated in a completely xenogeneic epithelial environment.     

Photoperiod and body weight in female Syrian hamsters: skeleton photoperiods, response magnitude, and development of photorefractoriness

Two experiments examined the effects of various photoperiods on body weight and reproductive function in Syrian hamsters (Mesocricetus auratus). In Experiment 1 female hamsters were exposed to symmetrical skeleton photoperiods in which dawn and dusk were mimicked by 1-hr light pulses. A skeleton long (LD 16:8) photoperiod had no effect on body weight or estrous cyclicity (compared with animals in a complete LD 16:8 photoperiod), but exposure to a skeleton short (LD 10:14) photoperiod increased body weight and interrupted estrous cycles. Thus, body weight appears to respond to the relative timing of the two light pulses (a circadian mechanism) rather than to the absolute amount of light or darkness (an hourglass mechanism), just as does reproduction. In Experiment 2 female hamsters were exposed to a long photoperiod (LD 16:8) or to one of two short photoperiods (LD 10:14 and LD 8:16). Both short photoperiods increased body weight and interrupted estrous cycles, but the LD 8:16 photoperiod was substantially more effective at increasing body weight than was the LD 10:14. Thus, hamster body weight appears to be capable of a graded response to photoperiod, with shorter days producing a greater obesity. With prolonged exposure to the two short photoperiods (greater than 30 weeks), body weights spontaneously returned to the same level as the long-day controls, and estrous cycles resumed. When these hamsters were treated with the pineal gland hormone, melatonin, only those housed in long days exhibited the characteristic body weight gains, growth of brown and white adipose tissues, and decreases in uterine weight. Therefore, with prolonged exposure to short days, energy balance develops a photorefractoriness and an insensitivity to melatonin treatment, just as with reproductive function.     

The bursal microenvironment: phenotypic characterization of the epithelial component of the bursa of Fabricius with the use of monoclonal antibodies.

Monoclonal antibodies were raised against newborn chick bursa of Fabricius, and here we describe two antibodies, BEP-1 and BEP-2, which react selectively with the epithelial component of the bursa of Fabricius. In previous studies, using quail chick chimeric bursas, we have demonstrated that the epithelium of the bursal rudiment, presumably of endodermal origin, gives rise to the epithelium lining the bursal lumen, the basement membrane-associated epithelium and the network of reticular cells of the medulla, while the interfollicular connective cells are derived from the mesoderm. When tested in indirect immunofluorescence assay on bursa tissue sections or cell suspensions, BEP-1 reacts with a surface antigen present on all the epithelial cells of the bursa and could be used as a marker for this cell lineage. BEP-2 binds to an intracytoplasmic antigen that is present in about 5% of cells, representing the epithelial cells, and which is excreted in the medulla. BEP-2 also reacts with the epithelial cells of the thymic medulla and with the mucin-secreting goblet cells of the intestinal villi. A rabbit antiserum raised against human cytokeratin gives a different pattern of reactivity on bursal tissue compared to BEP-1 and BEP-2, tentatively suggesting that these two antibodies do not bind to keratin-like molecules. During ontogeny, BEP-1 reactivity appears in bursal epithelium from the early stages of bursal ontogeny (8 days). BEP-2 reactivity is detected around hatching time. BEP-1 and BEP-2 do not show any antigenic heterogeneity among the epithelial cells of the bursa.     

Origin of the bursal secretory dendritic cell

The origin of vimentin-positive secretory dendritic cells of the bursa of Fabricius was studied by chick-quail chimera, parabiosis and immunohistochemistry using species-specific monoclonal antibodies. Quail bursal primordia of different ages were transferred to coelomic cavity of 3-day-old chicken embryos and further incubated for 18 days. In transplanted quail bursas the secretory dendritic cells of chicken and quail origin were detected by double staining of vimentin plus 74.3 and vimentin plus QCPN monoclonal antibodies, respectively. In bursal primordia of 5- and 6-day-old quail embryos both dendritic cells and B cells were of host, i.e. chicken origin. Mixed dendritic cell population of quail and chick origin emerged in chimeric birds of 6.5 days of age. In quail embryos transplanted at 7 and 8 days of age both dendritic cells and B cells were mixed i.e. of chicken and quail origin. Bursal secretory dendritic cells and medullary epithelial cells create "dendro-epithelial tissue" to receive pre-B cells. Colonization of dendro-epithelial tissue by pre-B cells initiates at day 7, thus the colonization of bursal anlage by blood-borne cells is a two-step process; entering of dendritic cells at day 6.5 is followed by that of B cells at day 7 and afterwards. It is discussed that bursal secretory dendritic cells and their product are key elements of bursal function therefore the mammalian bursa equivalent organ might be represented by a cell, which is analogous with the bursal secretory dendritic cell.     

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.     

Histochemical characterization of chicken lymphoid tissues

Thirteen enzyme activities were studied histochemically in the chicken bursa of Fabricius, thymus and spleen. The bursal epithelium contained a high activity of succinate, lactate and glucose-6-phosphate dehydrogenases and of ATPase. Activities of these enzymes were markedly lower in the follicle-associated epithelium than in the other parts of the bursal epithelium. A high activity of α-esterase occurred in the follicle-associated part in contrast to the other parts of the epithelium. The histochemical differentiation of the follicle-associated epithelium started at the time of hatching and was completed within a week. ATPase positive cells were observed in the interfollicular space of the bursa a few days before and after the hatching. The nature of these cells remained unclear. In the thymus, a good activity of lactate and glucose-6-phosphate dehydrogenases was observed in the area between the cortex and medulla, possibly indicating presence of endocrine cells. In the spleen no specific location of enzyme activities was observed.     

Development of the peripheral immune function in the chicken. A study on the bursa of Fabricius isolated from the rest of the gut-associated lymphoid tissue (GALT)

The bursa of Fabricius was surgically separated from the rest of the gut-associated lymphoid tissue (GALT). The antigens, sheep red blood cells (SRBC) and killed Brucella abortus organisms, were introduced into the bursal lumen, into the gut only, or allowed to get in contact with both (controls). The antibody titres in serum, the development of the bursal weight and the bursal histology were studied at various ages. The control birds responded vigorously to both antigens at between 2 and 8 weeks of age. At that age, the isolated bursa was capable of producing full anti-Brucella response, but not the anti-SRBC response, suggesting that SRBC need collaboration between both bursal and intestinal T and B cells. After 8 weeks, the cells in the gut alone were as potent as those in the gut and the bursa together at producing antibodies to SRBC. The bursal weight was significantly higher in the per bursam immunized chickens, indicating that continuous presence of antigens in the bursa may postpone its involution.     

Chemical bursectomy of chickens with colchicine applied to the anal lips.

To induce chemical bursectomy, 30 microliter colchicine dissolved in saline solution (1 mg/ml) was applied on the anal lips of White Leghorn chickens once daily for four consecutive days after hatching. Histologic characteristics of the bursa of Fabricius, spleen, thymus, cecal tonsils, and rectal wall were studied 1-7 days after hatching. Total necrosis of the lymphoid cells and the follicle-associated epithelium in the bursa was observed during the four days of colchicine application. The bursal stroma remained unchanged, and only minor changes were found in the interfollicular surface epithelium. After colchicine application ceased, some regeneration of the epithelium, as evidenced by small epithelial buds, was found. At the end of the observation period the epithelial buds were often covered by the follicle-associated epithelium, which was capable of phagocytizing carbon. However, practically no lymphoid repopulation was seen in the buds. Since this method of colchicine application had no direct effect on other lymphoid organs or on the survival or weight of the chickens, this bursectomy model seems to be a new tool for use in studies of bursal function.     

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.     

Cell transplantation into immunodeficient chicken embryos. Reconstituting capacity of cells from the yolk sac at different stages of development and from the liver, thymus, bursa of Fabricius, spleen and bone marrow of 15-day embryos.

Cyclophosphamide-treated 18-day-old chicken embryos were transplanted with histocompatible cells from the yolk sac at different stages of development and from the liver, thymus, bursa of Fabricius, spleen and bone marrow of 15-day-old-embryos. At the age of 36 days, the cell recipients were studied to determine the reconstitution capacity of the transplanted cells. The parameters used include the survival pattern, gain of body weight, antibody-forming capacity, response of peripheral blood lymphocytes to Con A, weight and microscopic morphology of the bursa of Fabricius, and weight of spleen and thymus. By all the criteria employed, only bursa cells were capable of a functional and morphological reconstitution of the recipient's humoral immune system. These data indicate that the role of the yolk sac as the first generator of prebursal stem cells remains questionable. In addition, these findings confirm the previous observations that, as a differentiation site of the B-cell lineage, the bursa of Fabricius precedes the bone marrow during ontogenetic development.     

Experimental infectious bursal disease in the ostrich (Struthio camelus)

Clinically severe disease was produced in ostriches aged 4 weeks by oral infection with a virulent strain of infectious bursal disease virus (vIBDV), namely strain Faragher 52/70. Four days after infection the birds were humanely killed and tissue samples, including thymus, bursa of Fabricius (BF), brain and kidney were collected for examination. Histopathologically, the thymus and BF showed severe lymphoid depletion and necrosis, while immunolabelling with a polyclonal antibody demonstrated abundant viral antigen.     

Comparison of pineal melatonin rhythms in young adult and old Djungarian hamsters (Phodopus sungorus) under long and short photoperiods

The pattern of the pineal melatonin rhythm was studied in 3-6- and 17-22-month-old female Djungarian hamsters under light-dark (LD) schedules, LD 16:8 and LD 8:16. Under both photoperiods, the amplitude of the melatonin rhythm was 2.5 times higher in young adult hamsters than in old ones. Two weeks after the change from LD 16:8 to LD 8:16, the period of elevated nocturnal melatonin levels lengthened by about 3 h in both age groups. It is suggested that old Djungarian hamsters, as well as young adults, might be able to recognize the length of a photoperiod and its change.     

Involution of the chicken bursa of Fabricius: a light microscopic study with special reference to transport of colloidal carbon in the involuting bursa

The involution of the bursa of Fabricius in White Leghorn chickens and the transport of colloidal carbon in the involuting bursa were studied on light microscopy. In chickens, from newly hatched to 23.5 weeks of age, the bursa was weighed, its histology was described, and the mitoses in the cortical and medullary compartments of the lymphoid follicles were counted. Decrease in bursal weight and in the number of mitoses were observed between 10 and 16 weeks of age. During week 17.5, the follicle-associated epithelium began to lose its endocytic capability, and mucin droplets appeared in the follicular medulla initiating the large mucoid cysts that were seen in the later phases of involution. The involutionary process was almost completed by week 23.5, when the bursa appeared as a fibrotic residue without intact lymphoepithelial structures. The particulate tracer used, colloidal carbon, was not observed to be transported to other organs from the bursa. The present study gives further support to the idea that after completing its central immune function in 6 weeks [21] the bursa, as estimated histologically, still possesses capacity to function like a peripheral lymphoid organ until 16 weeks of age.     

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.     

Ontogeny of spontaneous rosette-forming cells in the chicken. With special reference to the bursa of Fabricius.

Cells forming rosettes (RFC) with sheep erythrocytes (SRBC) and rabbit erythrocytes (RRBC) were studied during the ontogeny of the bursa of Fabricius of the chicken. The frequency of SRBC-RFC was low but significant in the bursa of 15-day-old embryos, and increased therafter in an approximately linear fashion with the age of the bursa donor until at least in 43-day-old chickens. The total number of SRBC-RFC per bursa increased throughout the ontogeny with no evidence for any abrupt increase after hatch. The mean number of SRBC bound per RFC also increased after hatch. In contrast the frequency of bursal RRBC-RFC was high in 15-day-old embryos, significantly decreased on embryonic day 18, remained low until at least 7 days after hatch and then increased significantly. The total number of RRBC-RFC per bursa in contrast remained relatively constant during the embryonic period and the first week after hatch and thereafter increased markedly. No change in the mean number of RRBC bound per RFC could be demonstrated during the ontogeny. RRBC-RFC were also frequent in the yolk sac, spleen, and thymus of 15-day-old embryos.     

Committed precursors of B and T lymphocytes in chick embryo bursa of Fabricius, thymus, and bone marrow

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.     

Effect of photoperiod on pineal gland volume and pinealocyte size in the chinese hamster,Cricetulus griseus

Male adult (200-day-old) Chinese hamsters (Cricetulus griseus) raised from weaning under either LD 16:8 or LD 8:16 were used. The pineal gland of the Chinese hamster consists of superficial (major) and deep (minor) components and a continuous, or interrupted, narrow parenchymal stalk interposed between them. The volume of the superficial pineal including the parenchymal stalk is greater under LD 16:8 than under LD 8:16. Under both photoperiods, pinealocytes in the superficial pineal have larger nuclei and more abundant cytoplasm than those in the deep pineal. Nuclei in the superficial pineal appear pale and usually have irregular profiles, whereas those in the deep pineal appear dark and have round profiles. In the superficial pineal, pinealocyte nuclei are larger, paler, and more irregular; and, in addition, nuclear density is lower under LD 16:8 than under LD 8:16. Similar, but less prominent, photoperiod-induced changes occur in the volume of the deep pineal, the size of pinealocytes, and pinealocyte nuclear morphology in the deep pineal. The results indicate that the development and differentiation of pinealocytes in both pineal portions may be advanced under long photoperiods and delayed under short photoperiods, although pinealocytes in the deep pineal may remain not fully differentiated even in adults. Since testicular weights and body weights are similar under both photoperiods, the photoperiod may exert marked influences on the development of the pineal gland without affecting reproductive activity and growth rates of animals.     

Immunoglobulin diversification in bursal duct-ligated chickens

The role of external antigen contact on immunoglobulin (Ig) diversification occurring in chicken bursal cells was evaluated. 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). We used two-dimensional gel electrophoresis to compare the heterogeneity of Ig molecules from bursa cells of normal and BDL chickens. We have found that Ig diversity obtained from BDL chickens' bursae in two-dimensional gel analysis was similar to that of control birds. Furthermore, by using two monoclonal anti-idiotype antibodies to study intrabursal Ig diversification we have shown that frequencies of the Cld-1 and Cld-2 idiotypes were also unaltered following bursal ligation. We conclude that primary B cell diversification in the bursa is independent of the external antigen flow from the bursal lumen.     

A study of peripheral tolerance through embryonic grafts of the bursal epithelial rudiment between MHC-distinct chick embryos

We report here that different organ grafts are not equally competentto induce tolerance of the host even if they are performed earlyin development, I.e. before the host's immune system has startedto develop. We have grafted the eplthello-mesenchymal rudimentof the bursa of Fabricius between hlstolncompatlble chickenembryos at E5 and found that the transplant is normally colonizedby hemopoletlc cells from the host and that a normal contingentof B cells is eventually produced. The grafted bursa is toleratedafter birth for a few weeks (4–8) but is in all casesrejected by an immune mechanism which cannot be assimilatedto the physiological involution process which occurs at 4–5months of age. Moreover, even in the first month after birth,when the bursa is still healthy, skin grafts of the same MHChaplotype are promptly rejected. These observations are in contrastwith the outcome of allogenelc limb bud grafts which are permanentlytolerated after birth although in an unperfect manner. We showin addition that, as was the case in xenogenelc grafts of limbbuds and bursas of Fabricius from quail and chick embryos, theallogenelc in situ graft to thymic epithelium of the MHC haplotypeof the bursal implant induces tolerance of the bursa. One commonpoint of xenogenelc and allogenelc embryonic grafts of limbbud, bursa and even thymic rudiments is that none of them induceda complete state of tolerance, since proliferation responseswere always obtained in vitro In one-way host-donor mixed leucocytecultures.