Chicken Thymus Research Articles

Lysis of Chicken Lymphocytes by Infectious Bursal Disease Viruses

Infectious bursal disease virus types 1 and 2 were able to induce direct lysis of chicken bursal cells, thymus cells, and peripheral blood lymphocytes in chromium-release assays. These two viruses were unable to lyse two established lymphoblastoid cell lines, although IBDV-1 was capable of multiplying in MSB-1 cells.

Alternative splicing within the chicken c-ets-1 locus: implications for transduction within the E26 retrovirus of the c-ets proto-oncogene.

Two overlapping c-ets-1 cDNA clones were isolated which contained the alpha and beta genomic sequences homologous to the 5' end of v-ets not detected in the previously described c-ets RNA species or proteins. Nucleotide sequencing demonstrated that these cDNAs corresponded to the splicing of alpha and beta to a common set of 3' exons (a through F) already found in the p54c-ets-1 mRNA. They contained an open reading frame of 1,455 nucleotides which could encode a polypeptide of 485 amino acids with a predicted molecular mass of 53 kilodaltons. However, when expressed in COS-1 cells, the cDNAs directed the synthesis of a protein with an apparent molecular mass in sodium dodecyl sulfate-polyacrylamide gel electrophoresis of 68 kilodaltons, p68c-ets-1, comigrating with a protein expressed at low levels in normal chicken spleen cells. These two proteins were shown to be identical by partial digestion with protease V8. Northern (RNA) blot hybridization analysis with the p68c-ets-1 -specific sequence and RNase protection experiments showed that the corresponding mRNA was expressed in normal chicken spleen and not in normal chicken thymus or in various T lymphoid cell lines. Thus, two closely related proteins, having distinct amino-terminal parts, are generated within the same locus by alternative addition of different 5' exons, alpha and beta or I54, respectively, onto a common set of 3' exons (a to F). Finally, we demonstrate that an aberrant splicing event between a cryptic splice donor site in c-myb exon E6 and the normal splice acceptor site of c-ets-1 exon alpha involved in the genesis of the E26 myb-ets sequence.

Comparative natural killer cell activities of thymic, bursal, splenic and intestinal intraepithelial lymphogytes of chickens

The natural killer (NK) cell activities of spleen, thymus, bursa, peripheral blood and gut intraepithelial lymphocytes (IEL) from FP and SC chickens were investigated in 4-hr and 16-hr 51Cr release assays. Target cells were 4 different tumor cell lines derived from either an avian leukosis tumor transplant (LSCC-RP9, LSCC-RP12) or from Marek's disease lymphomas (MDCC-MSB-1, MDCC-CU36), Great variability in cytotoxic potential was observed among NK cells of different lymphoid organs. NK cell cytotoxicity varied depending upon the type of effector cells, type of target cells, the ratio of effector to target cells, and the age and genetic background of chickens. Substantial levels of NK cell activity were detected in splenn and gut IEL of SC chickens in a 4-hr assay. In contrast, the NK cytotoxicity in gut IEL of FP chickens was not detectable until 16 hr after incubation. The ranges of target cell specificity demonstrated by IEL, spleen, thymus and bursa NK cells were similar to one another and, in general, the level of cytotoxicity increased with incubation time. Thymus and bursa NK cell activity of both SC and FP chickens was not detectable in a 4-hr assay but substantial NK cell activity was demonstrated in a 16-hr assay. The results of the present study demonstrate that various lymphoid organs of chickens, such as spleen, thymus, bursa, and gut intraepithelium, contain subpopulations of cells that can mediate spontaneous cytotoxicity.

Development of optimal conditions for lymphokine production by chicken lymphocytes

Chicken thymus, spleen, and bursa lymphocytes were isolated by different methods and incubated under differing conditions in order to obtain and characterize avian lymphokines. The biological activity of lymphokine—containing cell culture supernatants was measured by their antiviral activity (interferon(IFN)-units) and by their capacity to induce cytostatic effects in bone-marrow-derived macrophages (50 % cytostasis—inducing dose, CID). Lymphokine production by thymus lymphocytes required concanavalin A (ConA)—stimulation, while spleen cells, when cultured at high density, released CID and IFN activities into the culture medium even without mitogen-stimulation. By way of comparison, the highest lymphokine content was found in the supernatant of lymphocyte cultures, which were incubated for 72 hours at 41°C after stimulation with an optimal ConA dose. For stimulation of thymus lymphocytes 30 μg ConA/ml were found to be optimal, independent of serum content and cell density in the cultures. In contrast, the optimal ConA dose for spleen lymphocytes not only depended on the serum content but also on the cell density in the cultures and varied within a range of 2.5 μg and 45 μg ConA/ml.

Relationship between thymus ontogeny and DNA ligase expression in the chicken.

DNA ligase expression was studied in the embryonic chicken thymus. As previously reported, during thymus ontogeny two separate and successive (8S and 6S) forms of enzyme are found. The switch from the heavy to the light form takes place at around 18 days of incubation in situ. The demonstration that two successive generations of thymic lymphoid precursor cells develop in the embryonic bird thymus and that the replacement of the first by the second thymocyte generation takes place at around 18 days suggested the possibility that each form of enzyme could be restricted to a given thymocyte generation. By either culturing or transplanting embryonic thymuses of different ages it appears that the switch from the heavy to the light form of DNA ligase is not related to a change over of the first by the second generation of embryonic thymocytes but rather to some maturation process of the thymic lymphocytes starting from 17 days of embryonic life. The influence of extrathymic factors on the turning on of the expression of the 6 S DNA ligase in the post-natal environment is also suggested.

Lymphocytic depletion of bursa of Fabricius and thymus in chickens inoculated with Escherichia coli.

Specific-pathogen-free 10-week-old chickens were inoculated via the air sac with Escherichia coli and showed lymphocytic depletion of bursa of Fabricius and thymus. In experiment I, chickens were necropsied at 12 and 24 hours, 2, 3, and 5 days after inoculation. At 12 hours after inoculation there was lymphocytic depletion in the medulla of lymphoid follicles of the bursa. At 24 hours after inoculation there was lymphocytic depletion also in the cortex of follicles and edema in interfollicular interstitium and follicular medulla. At 2 and 3 days after inoculation there were more marked lymphocytic depletion in medulla and cortex, and fibrosis in interfollicular interstitium. Partial repopulation of follicles with lymphocytes was seen at 5 days after inoculation. In the thymus, lymphocytic depletion occurred in the cortex. At 12 hours after inoculation, lymphocytic necrosis increased in number more than that of control chickens. The width of the cortex and medulla decreased. At 24 hours after inoculation, lymphocytic necrosis increased further. At 2 to 5 days after inoculation, the boundary between the cortex and medulla of lobules was obscure and cellular elements of the cortex and medulla were mingled. In experiment II, chickens were necropsied as in experiment I and also at 8 and 14 days after inoculation. The relative weights of the bursa and thymus reduced rapidly to minimal relative weights at 8 days after inoculation. At 14 days after inoculation, both bursa and thymus had normal relative weights and histological structures. These findings indicate that E. coli infection may induce transient lymphocytic depletion of lymphoid tissues in the chicken.

Identification of c-myb (chicken), c-myb (mouse) and v-myb (AMV) protein products by immunoprecipitation with antibodies directed against a synthetic peptide

A synthetic nonadecapeptide (IL 19) derived from a sequence of v-myb was covalently bound to haemocyanin and used for immunization. Anti-IL 19 serum immunoprecipitated a 75 kDa protein in the lysate of metabolically labelled chicken and murine thymus cells. Presaturation of the serum with IL 19 abolished this immunoprecipitation, thus indicating that the product of c-myb in both chicken and murine thymuses is the 75 kDa protein (p75 c-myb). Anti IL 19 serum also precipitated p48 c-myb) in the lysate of nonproducer myeloblasts.

Detection of a New Antigen Associated With Chicken Thrombocytes on Marek's Disease Lymphoblastoid Cell Line<xref ref-type="fn" rid="FN2">2</xref>

A monoclonal antibody, 2C12, produced against MDCC-MSB1 cells, reacted with several cell lines derived from Marek's disease (MD), including MDCC-MSB1 cells, normal chicken thrombocytes, and MD tumor cells. It did not react with cell lines derived from avian leukosis or reticuloendotheliosis, cells from normal chicken thymus and bursa, chicken kidney cells and quail fibroblast cultures inoculated with MD virus, or with erythrocytes from 1-day-old and adult chickens, cow, sheep, and horse. Anti-thrombocyte serum prepared in a rabbit reacted with thrombocytes and MDCC-MSB1 cells. The specificities of antibody 2C12 and anti-thrombocyte serum against MDCC-MSB1 cells and thrombocytes were confirmed by cross-absorption, blocking, and double-membrane fluorescent antibody tests. The existence of a polypeptide with an apparent molecular weight of 103,000, common to both MDCC-MSB1 cells and thrombocytes, was demonstrated by Western blotting analyses.

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Interactions and quantitative analysis of immunoregulatory cells in the chicken thymus.

Step-wise dilution of chicken thymus cell suspensions has been used to sequentially reveal suppressor, effector, and helper cells in these suspensions. The cells were tested either alone or in autologous mixture combinations with peripheral blood lymphocytes (PBL) as a source of effector cells. The assays studied were graft-vs-host reaction (GvHR) and mixed lymphocyte (MLR) reaction, spontaneous cellular cytotoxicity and antibody-dependent cell-mediated cytotoxicity, and mitogen responsiveness to Con A, PHA, and PWM. When tested alone, high numbers of thymus cells (1 X 10(7) gave weak or low responses, with the exception of GvHR, which was high. When this number of thymocytes was mixed with a strongly responding PBL effector population, there was marked suppression of the latter. Nonspecific crowding was excluded as a cause for the decreased responsiveness, and the data therefore demonstrated the presence of suppressor cells in the thymus. With gradual reduction of the thymus cell number in the mixtures, the suppressor activity was lost, but concomitant with this was the appearance of, or a gradual increase in, thymus effector cells giving good responses. Further dilutions of the thymus (to, e.g., 1 X 10(5) cells) depleted the suspension of effector cells, but helper cells capable of markedly amplifying the effector potential of PBL were revealed. The suppressor/helper function of the thymus was not only dependent on the absolute numbers of thymus cells present, but also on the degree of inherent responsiveness of the effector PBL. If the response of PBL alone was strong, a thymus suspension containing both helper and suppressor cells (e.g., 1 X 10(6) cells) caused suppression of the PBL; if the PBL alone were weak, this same thymus cell suspension caused enhancement. The outcome of an immune response is therefore dependent not only on the presence or absence of particular cell types, but also on the ratios between these cells. An imbalance in these ratios in vivo may underlie diseases of immunologic origin, e.g., autoimmunity.

Isolation and characterization of chicken thymic electrolectin.

We have detected the presence of a beta-D-galactoside-binding lectin (electrolectin) in extracts of the thymus of adult chickens. This lectin was purified by affinity chromatography on a lactosyl-Sepharose column to yield 1.4 mg of pure protein from 230 g of thymus. The chicken thymic electrolectin (CTE) has an Mr of 15 300 when analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and of 30 000 when analysed by gel filtration. The amino acid composition of CTE is similar to that of other electrolectins purified from human and rat lung. CTE cross-reacts immunologically, but is not identical, with electrolectins from electric-eel electric organ and from chick-embryo pectoral muscle. CTE agglutinates chicken thymocytes but does not appear to promote their mitosis.

Effect of neuraminidase treatment on the expression of Fe 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.

Multiple sequence-specific DNA binding activities are eluted from chicken nuclei at low ionic strengths.

DNA sequence-specific binding proteins eluted from chicken erythrocyte and thymus nuclei, and fractionated as described by Emerson and Felsenfeld (19), have been investigated by filter binding and footprint analyses. The erythrocyte nuclear protein fraction specifically binds to at least two sites within the 5' flanking chromatin hypersensitive site of the chicken beta A-globin gene, and to a site 5' to the human beta-globin gene. The major chicken beta A globin gene binding site [G)18CGGGTGG) and the human beta-globin gene binding site [TA)6(T)8C(T)4) occur at or near sequences which are hypersensitive to S1 nuclease cleavage in supercoiled plasmids. Downstream, the second chicken beta A-globin gene binding site includes the beta-globin gene CACCC consensus sequence. Filter binding studies also show other sequence specific binding activities to human N-ras and human (but not chicken) c-myc gene sequences.

Activating effects of interferons, lymphokines and viruses on cultured chicken macrophages1

Cultured chicken bone-marrow-derived and blood monocyte-derived macrophages could be activated by various treatments: (1) With crude lymphokine preparations obtained from Concanavalin A (ConA)-stimulated chicken spleen or thymus cell cultures: (2) with virus-induced interferons (IFN) from cultured chicken embryo fibroblasts or macrophages; (3) with inactivated reovirus or live infectious bursal disease virus (IBDV). Macrophage activity was expressed by cytostatic effects against lymphoblastoid target cells, and by morphological changes, such as enhanced spreading of the cells. The macrophage-activating capacity of lymphokine preparations (50% cytostasis-inducing dose) was closely associated with their antiviral activity (IFN units). According to its physico-chemical properties, ConA-induced lymphocyte interferon was considered to be IFN-gamma, but acid-and heat-resistant IFN-alpha or IFN-beta may also have been present in spleen cell or thymocyte culture supernatants. Virus-induced interferons (IFN-alpha/beta) were less effective in macrophage activation than in antiviral activity. Experimental results strongly suggested that macrophage activation by viruses was mediated by endogenous IFN.

Quinolone antibiotics inhibit eucaryotic DNA polymerase α and β, Terminal deoxynucleotidyl Transferase but not DNA ligase

DNA polymerases α and β, Terminal deoxynucleotidyl Transferase and DNA ligases from chicken thymus were purified to homogeneity. Quinolone antibiotics (nalidixic acid, oxolinic acid and pefloxacin) known to inhibit DNA replication were tested for their effects on these enzymes. DNA ligase activity was not affected by the three drugs. DNA polymerases α and β were inhibited by competitive mechanisms. Surprisingly, Terminal deoxynucleotidyl Transferase was strongly inhibited by the three compounds and more efficiently by nalidixic acid. The significance of these results is discussed in terms of the possible involvement of the enzymes in the respective DNA replication and repair processes.

Monoclonal antibodies to human terminal transferase.

Human terminal deoxynucleotidyltransferase was used to produce mouse monoclonal antibodies. Three different assays were used to screen for terminal transferase antibodies and antibody-producing hybridoma cells: an enzyme binding assay, an enzyme-linked immunosorbent assay, and a "terminal transferase-specific antibody" cytochemical procedure that allows visualization of the homogeneity of the hybridoma clones producing antibodies to terminal transferase. Of the 12 mouse hybridoma clones isolated, seven produce IgG1 and five produce IgG2a immunoglobulins. Only one of these monoclonal antibodies is inhibitory to human terminal transferase activity. Four of the monoclonal antibodies react with terminal transferase peptides after separation on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, while the remainder appear to recognize only native determinants on the human enzyme. Cross-reactivity studies with purified calf thymus enzyme show that although four of the monoclonal antibodies bind to native enzyme, none react with calf thymus terminal transferase peptides after sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggesting structural differences within the cross-reactive determinants. A more general survey on the affinity of these monoclonal antibodies to human terminal transferase for the enzyme from other species using an enzyme-linked immunosorbent assay demonstrates that none of the monoclonal antibodies reacts with chicken thymus terminal transferase. The affinity of binding for terminal transferase from other mammals to this panel of monoclonal antibodies varies with the clones as expected, but few bind as strongly as the human enzyme. These results suggest that although antigenic determinants are conserved through evolution, fine structural changes within the determinants do occur.

Poly(A) polymerase distribution in normal and malignant lymphoid cells.

The incorporation of ATP on poly(A) primers catalyzed by poly(A) polymerase was investigated in normal and neoplastic lymphoid cells from animal and human sources. High levels of the enzyme were found in mouse thymus, in chicken bursa and thymus, as well as in neoplastic cells from patients affected by lymphoblastic and Burkitt's lymphomas. Low or very low quantities were found in peripheral blood lymphocytes, chronic lymphocytic leukemia cells, normal lymph nodes and solid lymphoid tissues of Hodgkin's disease. In general, the enzymatic content of neoplastic lymphoid cells reflected those of their normal counterpart. No effect of fasting or cortisone treatment on poly(A) polymerase in mouse spleen, thymus or liver was found. No particular relationships with B, T or non-T, non-B lineages were observed, but some relationship with DNA polymerase alpha was found. Therefore, it may be that poly(A) polymerase levels are related to the proliferative activity of the cellular populations.

MHC- and non-MHC-encoded surface antigens of chicken lymphoid cells and erythrocytes recognized by polyclonal xeno-, allo- and monoclonal antibodies.

Surface antigens on chicken thymus and bursa cells were analyzed by immunoprecipitation using polyclonal and monoclonal antisera raised against (and specific for) thymus (ATS) or bursa (ABS) cells, respectively. The antigens identified were compared with those governed by the B-F, B-L and B-G regions of the chicken major histocompatibility complex (B complex). Four proteins were precipitated from thymus cells by 2 polyclonal ATS: both antisera recognized molecules of apparent molecular mass of 172-182, 132-135, 75-76 kDa, and one antiserum in addition recognized a protein of 102 kDa. The 172-182 and 102-kDa peaks were still demonstrable under reducing conditions indicating that they are composed of a single polypeptide chain, the other 2 were lost under reducing conditions, therefore, must be composed of smaller subunits. Of the 2 monoclonal ATS tested, one identified a single protein of 186 kDa and the other a 135-kDa protein (in addition to 2 smaller molecules); whether these are the same as those precipitated by the polyclonal antisera remains to be determined as they behaved differently under reducing conditions. Proteins of 162 and 78-84 kDa were revealed by 2 polyclonal ABS under nonreducing conditions but the former may in one case be a polymer (it disappeared under reducing conditions) and in the other a single molecule. In addition molecules of 182 kDa were identified by one antiserum and of 84 and 60 kDa by the other under nonreducing conditions. Of the 4 monoclonal ABS only one identified a 200-kDa protein: molecules of 115-125, 90-100, 48-52 and 40-43 kDa were also precipitated, all of which were reduced to smaller molecules. With 2 specific anti-B-F alloantisera we were able to precipitate the "conventional" B-F antigen from red blood cell lysates of CB-strain chickens resolving into a 40-kDa peak and a light chain of about 12 kDa corresponding to beta 2 microglobulin. Precipitates from peripheral blood lymphocytes, bursa and thymus cells revealed an additional protein of 22 kDa. With 2 specific B-L alloantisera two peaks of 33 kDa and 31 kDa were obtained from peripheral blood lymphocytes. Using anti-B-G alloantisera a double band corresponding to 47 and 42 kDa was seen under reducing conditions. There is no evidence from these data to indicate that the polyclonal and monoclonal antibodies are directed towards major histocompatibility complex antigens.

Effect of infectious bursal disease on natural killer cell activity and mitogenic response of chicken lymphoid cells: role of adherent cells in cellular immune suppression.

A pathogenic isolate of infectious bursal disease virus (IBDV) caused persistent and extensive lesions in the bursa but mild and transient lesions in the thymuses of chickens of lines 63 and P. The effect of IBDV on two cellular immune functions, namely, natural killer cell cytotoxicity and mitogenic response, was studied. The natural killer cell activity was not consistently influenced, but the virus, during the first 2 weeks of infection, caused transient depression of the blastogenic response of spleen cells to phytohemagglutinin. Studies on mitogenic hyporesponsiveness revealed that the functional impairment was mediated by a suppressor cell that shared several characteristics with macrophages; i.e., the suppressor cell was adherent to plastic, was phagocytic, and resisted treatment with antithymocyte and antibursa cell sera. Removal of suppressor cells from the spleens of virus-infected chickens resulted in restoration of the mitogenic response of cells. Further, in mixing experiments, the suppressor cell isolated from the spleens of virus-infected chickens also inhibited the mitogenic response of normal spleen cells. We concluded that reduced mitogenic response of lymphocytes in IBDV-infected chickens was not due to a lack of functional T-cells, as suggested previously by others, but was due to macrophage-like suppressor cells. The suppressor cells, although present in certain normal chickens, became activated during early stages of IBDV infection.

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.