Identification of terminal N-acetylglucosamine residues of highly branched asparagine-linked oligosaccharides as immunoreactive domains of a chicken heterophile antigenic determinant

Chicken heterophile antigenic determinant (CHAD-1) has been previously found in medullary lymphocytes of the bursa and thymus as well as in some non-lymphoid cells by the immunoperoxidase method, using rabbit antiserum to a complete Freund's adjuvant (CFA) as the first antibody. In this work we demonstrated that absorption of anti-CFA serum with highly purified preparations of hen egg white glycoproteins (ovomucoid, ovoinhibitor, ovalbumin) or chicken orosomucoid completely blocked immunoperoxidase staining for CHAD-1. Treatment of these glycoproteins with beta-N-acetylglucosaminidase suppressed their capacity to inhibit this staining. Absorption of anti-CFA serum with asparagine-linked glycopeptides which have the mannose alpha 1,3 arm disubstituted by GlcNAc residues and which have another GlcNAc residue linked beta 1,4 to the beta-linked mannose of the core also inhibited staining for CHAD-1. These data indicated that highly branched asparagine-linked oligosaccharides with terminal GlcNAc residues beta-linked to mannose represent immunoreactive domains of CHAD-1.     

Specific antigens of chicken thymus

Purified plasma membranes from chicken thymus and bursa cells were prepared and solubilized with sarkosyl (sodium salt of N-methyl-N-(1-oxodecyl)-glycine). Antisera to solubilized thymus plasma membrane (TPM) were produced in rabbits and the globulin fraction obtained by ammonium sulfate precipitation. Four precipitating antigens were detected in solubilized TPM by immunoelectrophoresis. Following absorption with chicken serum and bursa plasma membrane (BPM) immunosorbents, three antigens, designated T₁, T₂, T₃, were specific for the TPM fraction, and one antigen, T₁, was found in soluble extracts of thymus tissue. Absorption with isolated plasma membrane and whole cells indicated that the T₁ and T₂ antigens in solubilized TPM are associated with the plasma membrane but not expressed on the surface of the cell. A common antigen, designated BT, was detected in BPM and TPM fractions and in membrane preparations of spleen. The antigens were not detected in any other tissues or cells including brain, circulating lymphocytes and erythrocytes.     

Lymphoid antigenic determinants of the chicken: ontogeny of bursa-dependent lymphoid tissue

The ontogenic development of the bursa-dependent lymphoid tissue in the chicken has been studied using rabbit antisera specific for B lymphocyte sub-populations and two elements of the bursal microenvironment. The antigens investigated were: the chicken B lymphocyte antigen ( CBLA ); mature B lymphocyte antigen ( CMBLA ); a foetal-associated antigen ( CFAA ) present on embryonic haemopoietic cells and adult bone marrow and bursa cells; immunoglobulin (Ig) and IgG; a bursa-specific cortical reticulin fibre antigen ( CBRFA ); a gut-associated mucin antigen ( CGAMA ) present on bursal medullary reticular epithelial (REp) cells. The development of suspected precursor cells was examined using a rabbit antiserum specific for foetal spleen cells. The major finding was the interrelationship between developing B cells and the bursal microenvironment. The first CBLA - and CFAA -positive cells were detected in the bursa at day 8 of incubation but their precise localization was difficult to assess. In 12-15 day embryos, both these cells were found predominantly in the tunica-propria in close proximity to cells bearing the reticulin fibre antigen CBRFA . This close association between CBLA -, CFAA - and CBRFA determinants represents the earliest stages of B cell differentiation and mimics that found in the adult bursa cortex. By day 18, the majority of bursa cells expressed CBLA and Ig and were localized in the developing medullary follicles, the REp cells of which were CGAMA -positive, demonstrating a very early interaction between intestinal tract contents, bursal REp cells and B cell maturation. Around hatching some bursa cells showed a marked increase in the membrane expression of CBLA and Ig, and the simultaneous expression of CMBLA and IgG. These cells were present in medullary follicles; CBRFA was present on cortical reticulin fibres which provided a supporting framework for the more immature CFAA -positive cells.     

Bu-2, a novel avian cell surface antigen on B cells and a population of non-lymphoid cells, is expressed homogeneously in germinal centers

A monoclonal antibody (mAb), Hy30, was generated against bursal cells. Under reducing conditions, it immunoprecipitates a cell surface antigen with an apparent molecular weight of 66 kD that is distinct from immunoglobulin and MHC class II. The antigen recognized by Hy30 is found on greater than 95% of all bursal cells, on a population of thymus resident cells, and on blood, spleen, and marrow mononuclear cells. Immunohistological analysis of tissue sections revealed that the majority of the staining by Hy30 occurs in the B cell areas of the spleen and thymus. Flow cytometric analysis demonstrated that the antigen recognized by Hy30 is found on both immunoglobulin positive (Ig+) and negative (Ig-) cells. Analysis of a chicken rendered B cell deficient by treatment with cyclophosphamide at hatch confirmed this observation. In these birds, the medullary dendritic cells of the bursa and Ig- cells of the "B-cell areas" of the spleen and thymus expressed the Hy30 Ag. Analysis of several avian tumor lines did not suggest an obvious lineage for these Hy30+ Ig- cells. However, analysis of germinal centers suggested that these cells may be tissue macrophages or dendritic cells. This is consistent with previously reported histology of these "B-cell areas". Flow cytometric analysis of the antigen recognized by Hy30 demonstrates that it is distinct from the Bu-1 B-cell surface antigen and we designate it as Bu-2.     

Solubilization and partial purification of lymphocyte specific antigens in the chicken

Four different lymphocyte antigens were solubilized from chickens homozygous at the B locus. The antigens were identified by immunoelectrophoresis with rabbit anti-lymphocyte sera. 1. A bursa-specific cell surface antigen was extracted with 3 M potassium chloride and partially purified. The antigen was excluded from Sephadex G-200 gels, was heat labile and was a potent immunogen. The antigen could be detected on all bursa cells by immunofluorescence but not on thymus, spleen or blood lymphocytes. The findings do not exclude, however, the possibility that a small number of lymphocytes (e.g. plasma cells) possess the antigen. 2. A thymus-specific antigen was obtained by papain treatment of thymus cells. It migrated cathodically in immunoelectrophoresis. 3. A surface antigen which was specific for lymphocytes from both central lymphoid organs (thymus and bursa) was solubilized by pestle homogenization. 4. A fourth lymphocyte surface antigen was present on lymphocytes from blood, spleen, bursa and thymus. It was best solubilized by 3 M potassium chloride extraction and did not migrate under the conditions of immunoelectrophoresis. Studies did not reveal a stable antigenic marker with specificity for thymus or bursa cells and their progeny in the peripheral lymphocyte pool.     

Pathogenicity of chicken anaemia virus (isolate 10343) for young and older chickens.

One-day-old chicks, inoculated intramuscularly (i.m.) with the chicken anaemia virus (CAV) isolate 10343, showed depression of body weight gain and anaemia, particularly between days 14 and 21 post-inoculation (p.i.)- The weights of thymus and bursa were substantially reduced compared to controls at days 14 and 21 p.i. The histological lesions detected in thymus, bursa, spleen and liver were similar in frequency at days 14 and 21 p.i. Eosinophilic intranuclear inclusion bodies, lymphocyte depletion, and focal necrosis were detected in the thymus, spleen, bursa and liver of more than 50% of the inoculated chicks at days 14 and 21 p.i. Focal necrosis and vacuolar degeneration in the liver, as well as apoptosis in different organs were more evident at days 14 and 21 p.i. Ten-week-old broiler breeders, inoculated i.m. with isolate 10343 showed pathological changes that were less severe than the changes shown by 1-day-old chicks. No anaemia could be detected in this group. However, severe thymus atrophy, and histological lesions in bursa, spleen, and liver, were also evident at days 14 and 21 p.i. in some of the inoculated birds. Viral detection by immunofluorescence using a monoclonal antibody revealed a wide distribution of the CAV isolate. CAV antigen was detected until day 21 p.i. in thymus, spleen, bursa and liver. According to the severity of the lesions shown by 1-day-old chicks, the length of the period in which CAV antigen could be detected in tissues, and the fact that CAV isolate 10343 was capable of inducing disease in 10-week-old chickens, it seems that this CAV isolate may be particularly virulent.     

Chicken immunoregulatory Ig-like receptor families: An overview and expression details on ggTREM-A1

Paired immunoregulatory receptors facilitate the coordination of the immune response at the cellular level. In recent years, our group characterized chicken homologues to mammalian immunoregulatory Ig-like receptor families. The first part of this review focuses on the current progress on chicken immunoregulatory Ig-like receptor families. One of these receptors is gallus gallus TREM-A1, which was described as the only member of the chicken TREM family with activating potential. The second part of this review presents a study initiated to further characterize ggTREM-A1 expression. For this purpose we established real-time RT-PCR and generated a specific mab to analyze the expression profile of ggTREM-A1 on mRNA and protein level, respectively. GgTREM-A1 mRNA was predominantly expressed in macrophages, but was also detected in brain, bone marrow, bursa, thymus, spleen and PBMC. Analyzing ggTREM-A1 surface expression by mab staining validated the expression on macrophages. Additionally, we showed high expression on blood monocytes, heterophils and NK cells and on monocytes isolated from bone marrow. Moreover, we detected ggTREM-A1 protein also on thrombocytes, B and T cell subsets, but antigen expression seemed to be lower and more variable in these cells. Immunohistochemistry of chicken brain tissue, combining ggTREM-A1 mab and various markers specific for various brain cell subsets showed expression of ggTREM-A1 on microglial cells, but also on neurons, astrocytes and oligodendrocytes. In conclusion, ggTREM-A1 is expressed on a variety of cells, relevant for the immune system, possibly combining physiological function of different mammalian TREM.     

The influence of bursocytes and thymocytes on graft versus host reaction and IgG level in chickens.

Graft versus host reaction was induced by the blood of chickens previously transferred by the bursa and thymus cells. These bursa and thymus cells were taken from bursectomized or thymectomized chicken. It was occurred that graft versus host reaction was influenced by transfer of bursa and thymus cell. IgG level was affected by the transfer of these cells too.     

Abnormalities associated with the bursal microenvironment in spontaneous dysgammaglobulinemia of UCD line 140 chickens

UCD line 140 chickens have been previously reported to develop a syndrome of spontaneous 7s immunoglobulin deficiency and the presence of autoantibodies. Earlier studies demonstrated that these inbred birds have normal peripheral blood T and B cell numbers; they also respond normally to allogeneic stimulation. Although the 7s immunodeficiency does not manifest itself until several months of age, line 140 birds have a premature degeneration of bursa. Because of the recent development of monoclonal reagents specific for bursal elements, including surface epithelium, basement membrane associated epithelium, follicle associated epithelium, and lymphoid subpopulations, we have examined line 140 and control birds for the expression of bursal epithelial cell antigens. Line 140 birds, in contrast to control chickens, have a dramatic early alteration in the expression of an epithelial cell marker in the bursa, thymus, and intestine. Moreover, to further address this issue, we transplanted bursa from 10-day embryos onto the chorioallantoic membrane, a privileged site. Bursae from control birds became abnormal when transplanted onto line 140 CAM; they remained normal when transplanted among several control chicken lines. In contrast, line 140 bursa remained abnormal independent of the transplant procedure. Due to the marked bursal abnormality observed specific to the dysgammaglobulinemia chicken line, we propose that the microenvironmental features of line 140 bursa may predispose these birds to the development of humoral immunodeficiency and autoantibodies.     

Bursa lymphocytes and IgM-containing cells in chicken embryos bursectomized at 52-64 hours of incubation

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.     

Effect of neuraminidase treatment on the expression of Fc IgG receptors on chicken embryonic bursa and thymus cells

Bursa and thymus cells from chicken embryos at different ages were analyzed for Fc IgG receptors by EA-rosette technique and by binding of heat aggregated chicken IgG (agg IgG) in the indirect immunofluorescence test. Neuraminidase treatment resulted in a substantial increase of agg IgG binding cells both in the embryonic bursa and thymus. The binding of agg IgG was shown to be specific for Fc IgG receptors, since IgM and F(ab')2 fragments were not bound to neuraminidase-treated embryonic cells. A far lower percentage of EA-rosette-forming cells than agg IgG binding cells were found both in untreated and neuraminidase-treated bursa and thymus cells. It was concluded that neuraminidase can reveal additional Fc IgG receptor sites mainly for agg IgG on embryonic thymus and bursa cells.     

Ontogeny and tissue distribution of the chicken Bu-1a antigen.

By using a sensitive technique of immunofluorescence on polyethylene glycol-embedded tissue sections, we could better determine the distribution of L22+ cells in embryonic and adult chickens. L22 mAb was originally described as reacting with bursa and bursa-derived lymphocytes. We now present evidence to suggest that this mAb also reacts with a subset of macrophages found in bursa, thymus, spleen, liver, intestine and peritoneum. The L22+ cells appear early during embryonic life, simultaneously in yolk sac, bursa, thymus, spleen and bone marrow. At all steps of their ontogeny, thymocytes were L22-, while most, if not all, bursal lymphoid cells were L22+. Moreover, L22 antigen can be detected on haemopoietic cells probably precursors, before and during their entry into the bursal rudiment on Day 9 or 10 of embryonic life. We conclude from these data that L22 is not restricted to the B-cell lineage as it is shared with a subset of macrophages. Furthermore, our observations of L22+ cells during embryonic life favour the hypothesis of separate lineages for B-cell and T-cell precursors in chick embryo, which was previously put forward on the basis of different sets of experiments.     

Mitotic rate maturation in the Peyer's patches of fetal sheep and in the bursa of Fabricius of the chick embryo

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.     

Chicken thymocyte antigen which participates in cell proliferations of thymocytes and tumour-derived lymphoid cell lines

A novel antigen on chicken thymocytes was defined by CETHB1 and CETH46 monoclonal antibodies (mAbs) that were prepared against chick embryonic thymocytes. CETHB1 and CETH46 mAbs recognized different epitopes on the same membrane antigen (molecular weight: 76.2 kDa). These mAbs reacted with >80% of thymocytes of 14-day-old embryos to 8-week-old chickens. Almost all splenocytes, peripheral blood lymphocytes and bursa cells were negative, and only 7.7% of bone marrow cells were positive for both antibodies. In two-colour analysis with mAbs reacting to T cell markers (CD4 or CD8), most CETHB1 positive cells were CD4 (- )CD8 (-) or CD4 (+) CD8 (+) . However, a proportion of CD4 (+) CD8 (-) and CD4 (- )CD8 (+) cells were negative for CETHB1 mAb. The proportion of thymocytes reacting with CETHB1 in chickens immunosuppressed by cyclophosphamide treatment increased gradually in parallel with the restoration of the thymus. An increase of CETHB1-positive cells was observed in thymocytes stimulated with Con A. Hence, it seems that the CETHB1 antigen expression on thymocytes is influenced by the thymic micro-environment and that the antigen may take part in thymic differentiation. Interestingly, CETHB1 antigen was expressed not only on T cell tumour-derived lymphoid cell lines, but also B-lymphoma-derived cell lines. The antigen expressions on these cell Unes were observed only in the prohierative phase of the cells. Hence, the molecule which reacted with CETHB1 may be an antigen commonly expressed on lymphoma cells and may be involved in cell proliferation.     

Ontogeny and function of two non-lymphoid cell populations in the chicken embryo

The purpose of the study was to determine what type of non-lymphoid cells develop in chicken embryos during ontogeny, and whether these cells are functional. To detect these cells, we used monoclonal antibodies specific to two groups of non-lymphoid cells: CVI-ChNL-68.1, specific for mononuclear phagocytes, and CVI-ChNL-68.2, specific for a subpopulation of reticulum cells in spleen, liver and bursa. Monoclonal antibodies HIS-C7, HIS-C1, and HIS-C12, which are specific to leukocytes, B lymphocytes, and IgM respectively, were used to correlate the ontogeny of non-lymphoid cells and lymphoid cells. Mononuclear phagocytes and reticulum cells were detected in the liver, spleen, yolk sac, bursa, gut, and thymus at about the same time as leukocytes, but earlier than B lymphocytes. To determine whether mononuclear phagocytes and reticulum cells in spleen and liver absorb antigen, we injected embryos intravenously with colloidal carbon and the antigen FITC-Ficoll. In addition, acid phosphatase was used as a marker for phagocytic activity. Reticulum cells in the liver and spleen were functional from the first point of detection, whereas mononuclear phagocytes in the liver and spleen started to absorb antigen a few days after their development.     

个体发育中北京鸭胸腺细胞的抗原及其在部分禽类胸腺与脾中的分布

从1日龄北京鸭胸腺细胞免疫的兔抗血清经适当吸收后,与鸭脑无交叉反应。反之,用鸭脑匀浆免疫的兔抗血清也不与鸭胸腺细胞起交叉反应,初步结果表明鸭脑与胸腺细胞上无共同抗原。该抗血清与鸭哈德尔氏腺细胞不起反应。抗鸭胸腺血清与鸭、来杭鸡与日本鹌鹑的胸腺细胞起交叉反应,但与鸽、小鼠、豚鼠的胸腺细胞不起反应表明是针对部分禽类胸腺细胞上的共同抗原决定簇。就个体发育中的鸭胚胸腺和脾细胞上的分布进行研究的结果表明该抗原最早出现在14天的鸭胚胸腺细胞和15天的鸭胚脾细胞上。     

Cytoplasmic CD3+ surface CD8+ lymphocytes develop as a thymus-independent lineage in chick-quail chimeras

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.     

Studies on antilymphocyte antibody in the chicken. II. Effect of antithymus and rabbit antibursa globulin on immune reactions in young chickens

Four-week-old chickens repeatedly injected intraperitoneally with heterologous antithymus globulin (ATG) and antibursa globulin (ABG) were immunized, in the course of treatment, with bovine γ-globulin (BGG). The ATG and ABG depressed the production of anti-BGG antibody. On the other hand, only ATG was effective in suppressing experimental allergic encephalomyelitis. Immunoelectrophoretic analysis of sera from chickens treated with ATG, ABG and NRG (normal rabbit globulin) revealed that those globulins are themselves immunogenic. ATG and ABG induced in the spleen a moderate depletion of lymphocytes and plasma cells respectively. These reagents produced lymphocytopenia and granulocytopenia, but a similar, although less expressed, effect on leucocytes in the peripheral blood could be induced by NRG. Thymus, bursa, caecal tonsil, Peyer's patches and lymphoid masses of the intestine were not influenced by ATG, ABG and NRG. The results are discussed and interpreted as further evidence for the delineation between immune functions of the thymus and the bursa of Fabricius in the chicken.     

Identification of a subpopulation of chicken B lymphocytes by the lectin from Lotus tetragonolobus

Lectin-reactive chicken lymphoid cells were detected by agglutination. The lectin from Lotus tetragonolobus agglutinated cells from bursa and spleen and did not agglutinate cells from thymus or peripheral blood of 28-day-old chickens. The percentages of Lotus tetragonolobus reactive cells were also measured by assays of lectin-induced rosette formation, binding of lectin-labelled latex beads, and binding of rhodamine-labelled lectin. The distribution of lectin reactive cells varied with the age of the chicken. The lectin appears to identify a unique subpopulation of chicken B lymphocytes.     

Susceptibility of thymocytes for infection by chicken anemia virus is related to pre- and posthatching development.

To investigate the age-dependent mechanism of susceptibility for chicken anemia virus (CAV) infection, we inoculated embryos and chickens of ages between day 9 of embryonic development and day 28 after hatching with CAV. Chicken embryos inoculated at days 9 and 11 of development showed no CAV-infected cells in the thymus, nor in other lymphoid organs. Many CAV-infected cells were detected in the thymic cortex of all chicken embryos inoculated at days 13 and 16 of development and of all chickens inoculated 1, 3, and 7 days after hatching. All embryos and chickens that contained CAV-infected cells in the thymus also contained CAV-infected cells in the bone marrow, but not in the bursa of Fabricius or the spleen. In chickens inoculated at days 14 and 21, only few CAV-infected cells were detected in the thymus, whereas these cells were not detected in thymi of 28-day-old inoculated chickens. Depletion of the thymic cortex was only detected in chickens inoculated from day 16 of embryonic development till day 21 after hatching. Only hematocrit values of the chickens inoculated 1 and 3 days after hatching were below normal. The rationale for the simultaneous susceptibility of cells of the T-cell lineage and cells of the erythrocyte lineage is discussed. As far as the thymus is concerned, the absence of clinical and microscopical signs of CAV infection in older chickens and the inability of CAV to infect embryos at days 9 and 11 of embryonic development may be caused by a lack of susceptible thymocytes.(ABSTRACT TRUNCATED AT 250 WORDS)