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Cytosolic Ca(2+) is known to be an important factor in intracellular signaling pathways that regulate several cellular functions. The present study was designed to measure the intracellular concentrations of Ca(2+) ([Ca(2+)](i)) in T cell mitogen-stimulated chicken lymphocytes, and to compare the results with those in rat lymphocytes. [Ca(2+)](i) was increased in the thymocytes, splenocytes and bursacytes of chickens, and in the thymocytes and splenocytes of rats following exposure to the mitogens phytohaemagglutinin (PHA) and concanavalin A (ConA). Increases were greatest in the thymocytes followed by the splenocytes and bursacytes. The PHA-induced changes in the thymocytes and splenocytes were similar in chickens and rats, but the ConA-induced increases were significantly lower in the chickens than rats. Pretreatment with EGTA before the application of PHA and ConA completely suppressed the rise in [Ca(2+)](i) in all the chicken lymphocytes, indicating that the increases that occurred in PHA- and ConA-treated chicken lymphocytes could be entirely attributed to the influx of extracellular Ca(2+). On the other hand, the PHA- and ConA-induced increase in [Ca(2+)](i) in rat lymphocytes was not completely suppressed by EGTA, indicating the recruitment of Ca(2+) from the intracellular Ca(2+) pool. The results suggest species differences in the Ca(2+)-based responses to T cell mitogens between chicken lymphocytes and rat lymphocytes.

The Ets-1 proto-oncogene is a prototype member of Ets family of transcription factors. It is preferentially expressed in lymphoid cells, where it is essential for the maintenance of the normal pool of resting T and B cells. We have investigated the protein expression of the Ets-1 transcription factor during the activation and apoptosis of T and B cells by flow cytometry and confocal microscopy. Cells of the thymus, spleen and bursa expressed high levels of Ets-1 protein, while resting peripheral blood mononuclear cells had lower Ets-1 expression. Activation and proliferation of T cells induced the upregulation of Ets-1. alphabeta-T cells were found to upregulate Ets-1 expression more than gammadelta-T cells. Increased Ets-1 protein expression was located predominantly in the perinuclear area. In contrast, during apoptosis, Ets-1 expression was downregulated. Collectively, our results indicate that Ets-1 expression can be accurately determined by flow cytometry and confocal microscopy. Ets-1 expression level and distribution are differentially controlled in resting, activated and apoptotic lymphocytes.

Mallard ducklings were fed diets containing corn naturally contaminated with mixed aflatoxins, purified T-2 toxin, or no detectable mycotoxin in two trials. The aflatoxin level used was 12 ppb in the first trial and 33 ppb in the second. T-2 was added at 2 ppm in both trials. No pathology was associated with the aflatoxin used in this study, and T-2--induced lesions were described in a previous publication. The weights of primary (thymus and bursa of Fabricius) and secondary (spleen) lymphoid organs were significantly reduced in the T-2--treated birds. The total number of viable cells recovered from the thymus was significantly reduced in aflatoxin-treated birds. The numbers of viable cells recovered from thymus, bursa of Fabricius, and spleen were all significantly reduced after treatment with T-2. In each trial, significantly lower mitogenic responses were seen to pokeweed mitogen and concanavalin A in birds fed aflatoxin or T-2, representing reduction in both B-cell and T-cell mitogenesis. Birds fed aflatoxin also had significantly reduced Escherichia coli O55 lipopolysaccharide-induced mitogenic responses. These studies indicate that subacute oral exposure to aflatoxin caused a loss of normal lymphocyte reactivity in mallard ducklings. This finding supports the hypothesis that waterfowl that ingest even small quantities of mycotoxin-contaminated waste grain are likely to be more susceptible to bacterial or viral infections.

Background: The so-called diffusely infiltrated lymphoid tissue of the chicken bursa of Fabricius was previously described as a T-dependent bursal area., Methods: We have analyzed immunohistologically its postnatal development by using a battery of mAbs, most of them raised specifically to chicken T cells, in order to characterize phenotypically the nature of its cell components, appearance, development, and possible functional significance., Results: Our results demonstrate that this tissue represents poorly developed lymphoid aggregates, the cell content reminiscent of that found in other lymphoid tissues occurring throughout the avian gut. The first lymphoid elements appear in this bursal area only after hatching, growing rapidly to reach the adult condition in the second week of postnatal life. They consist mainly of T lymphocytes, including principally. CD4+ TcR alpha V beta 1+ cells, which form cell groups and CD8+ cells, and TcR gamma delta+ lymphocytes, which predominate in the subepithelial area and within the epithelium. MHC Class II molecule-expressing cells and IgM-, IgG-, and IgA-positive B lymphocytes also occupy the subepithelial region., Conclusions: We propose that the bursal diffusely infiltrated lymphoid tissue of the chicken represents gut-associated lymphoid tissue involved in mounting immune responses to antigens that reach the bursal lumen via the cloaca.

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.

Bursocytes and thymocytes of chickens previously subjected to surgical removal of thymus or bursa Fabrici or to rtg irradiation, were analyzed for the level of cyclic nucleotides. As observed, regardless of the origin of the lymphocytes cells, cAMP was quantitatively prevailing; cGMP amount was smaller in most cases. As for the absolute values, the level of cyclic nucleotides was highest in bursocytes and lowest in peripheral blood lymphocytes (control). Biological consequences of these relations were discussed.

Sex steroids were found to affect both murine and avian immune systems. Female and male (NZB X NZW)F1 mice were castrated at 2 weeks of age and given Silastic implants containing either dihydrotestosterone or estradiol. Four weeks following treatment, the thymuses were studied for cell cycle kinetics and for the presence of various cell surface antigens using fluorescein-conjugated antisera and flow cytometric techniques. Estradiol therapy resulted in an increase in mature thymocytes, that is, thymocytes that had decreased peanut agglutinin receptors and decreased Thy 1 antigens on their surfaces. Additional studies with anti-Lyt 1 and 2 indicated that these mature thymocytes were of the "helper" rather than the "suppressor" phenotype. Estradiol therapy resulted in an increase in the percentages of proliferating cells in the spleen and a decrease in the percentages of proliferating cells in the thymus. In contrast, in the avian system, estradiol had little effect on proliferation in immunological organs. Dihydrotestosterone was a potent inhibitor of proliferation in the avian bursa. These results indicate that sex hormones have specific effects on different immune cell subpopulations. In the murine system the male is the heterogametic sex but in the avian system the female is the heterogametic sex. Based upon the present and previous studies, we suggest that the primary modulating hormone for immunological sex effects in the mammal is estrogen, whereas in the bird it is androgen.

Utilizing the acid alpha-naphthyl acetate esterase histochemical staining technique, cells with established T-cell (thymus-derived) staining characteristics could be consistently demonstrated in the periarteriolar lymphatic sheaths of the spleen and the diffusely infiltrated area of the cloacal bursa. The same areas failed to stain positively in significant numbers following a preincubation period with a specific inhibitor for carboxyl esterase. Lymphoid cells in the thymus, the splenic follicles, and bursal follicles were mostly negative.

MHC class-I antigens on peripheral blood macrophages and bursa cells were analysed after adoptive bursa cell transfer to cyclophosphamide-treated immunodeficient chickens. We studied the expression of B-F locus-encoded antigens (B-F is homologous to mouse H-2K, D) on macrophages and B cells and found that macrophage B-F antigens are of host origin, whereas bursa cells express only donor-type B-F antigens. Both syngeneic and allogeneic bursa cells restored IgM antibody production to a T-cell-independent antigen, Brucella, but only syngeneic bursa cells could restore the IgG antibody response to a thymus-dependent antigen, sheep erythrocytes. The results indicate that B-cell maturation and macrophage-B-cell interaction are not MHC-restricted but that a restriction exists in T-cell-B-cell collaboration.

The role of chicken bursal and thymic reticular epithelial cells (REp cells) in the induction of B- and T-lymphocyte differentiation has been investigated using in vitro culture of these cells. Splenic adherent cells were used as controls. Precursor populations were obtained predominantly from embryonic bone marrow or spleen and incubated with REp cell monolayers for 72 hr. Differentiation was assessed by the expression of specific membrane antigens: chicken B lymphocyte-specific antigen (CBLA) and chicken T lymphocyte-specific antigen (CTLA). Bursal REp cells specifically induced approximately 20-30% of the embryonic cells to express CBLA but had no influence on CTLA expression. Conversely, CTLA, but not CBLA, was induced in approximately 20% of the embryonic cells by the thymic REp cell cultures. No effect on the expression of either antigen was observed with the splenic adherent cells. The induced cells correspond to the first stage of B- and T-lymphocyte differentiation found in vivo. The assay system also facilitated the study of shifts in precursor content of various organs with development. Whereas embryonic bone marrow of all ages studied contained both B- and T-lymphocyte precursors, these were not demonstrable in donors after hatching. Use of 12-day embryonic bursa and thymus suspensions as sources of stem cells indicated that precommitment to the B- or T-lymphocyte lineage is irreversible.

In order to study the bursal origin of T cells, 18-d chromosomally-marked bursa cells were transplanted into 18-d chick embryos treated with cyclophosphamide (CY). Transfer of bursa cells restored not only the humoral immune functions but also the mitogenic responses of peripheral blood lymphocytes against phytohaemagglutinin and concanavalin A (Con A). Transplanted bursa cells proliferated primarily in the recipients' bursa. Only 8 out of 209 Con A responsive spleen cells were of donor origin, but no donor-derived cells were observed in the thymus. These findings suggest that cells in the 18-d embryonic bursa are already committed to the B cell lineage, and are not capable of migrating into thymus and of developing into mature T cells.

The detailed study of the genetic control of T-B cell interactions in the chicken has been hampered by the lack of defined major histocompatibility complex (MHC) recombinant chicken lines. In the present study we have used some recently described MHC recombinant chicken lines separating regions encoding antigens that are homologous to class I and class II antigens of mammals in adoptive bursa cell transfer experiments, in which bursa cells from newly hatched chicks were transplanted into cyclophosphamide (Cy)-treated chicks. Subsequent immunizations of the recipients with a thymus-dependent antigen (SRBC) and a thymus-independent antigen (Brucella abortus) showed that the generation of germinal centers in the spleen and the production of antibodies to SRBC required identity between donor and recipient class II antigens (B-L antigens), whereas response to Brucella antigen did not require identity at any of the known MHC loci of the chicken. The results thus reveal that also in the chicken class II (B-L) region genes encode cell-surface glycoproteins that serve as restriction elements in T-B cell cooperation.

Transplantation of bursa cells into surgically thymectomized (STx), cyclophosphamide(CY)-treated and X-irradiated (X) newly hatched chicks resulted in a morphological and functional reconstitution of the bursa-dependent immune system only when the transplanted bursa cells were supplemented with thymus cells. Without supplementation with thymus cells, morphological restoration of the bursa, antibody formation to sheep red blood cells and Brucella abortus, and germinal center formation remained deeply deficient. Bursa cells alone induced a full reconstitution of the bursa-dependent immunity in non-thymectomized. CY-treated and X-irradiated cell recipients. The incomplete restoration of STx-CY-X chickens after transplantation of bursa cells was not due only to lack of T helper cells, as indicated by the incomplete restoration of the bursal morphology and of antibody formation against Brucella abortus, a thymus-independent antigen. It is concluded that T cells are necessary for the B cell differentiation in the bursa of Fabricius. The mechanism behind this phenomenon remains unclear but our results indicate that a complete T cell function is not a prerequisite for this, since STx-CY-X birds reconstituted with bursa and thymus cells together did not have their mitogen (PHA, Con A) responses normalized.

Within the cloacal bursa (bursa of Fabricius), there is a diffusely infiltrated area (DIA) of lymphoid cells just dorsal to the bursal duct opening. Because this region displayed certain histologic characteristics of thymus-dependent areas found in mammals, such as high endothelial venules, heavy reticular fiber components, and epithelium infiltrated with lymphocytes, experiments were conducted to determine whether thymus-derived cells (T cells) were present. Two independent methods substantiated the presence of T cells in the DIA. After the acid alpha-naphthyl acetate staining technique, the characteristic T cell focal esterase activity could be demonstrated in significant numbers of cells found in the DIA. Also autologous radioactively labeled thymocytes could be identified in the DIA by autoradiography a few hours after IV injection. This establishment of a definite T cell area within the bursa gives additional credence to the contention that the bursa functions as a secondary lymphatic organ.

Cultures of chicken lymphoid tissues were tested for their capacity to lyse (51)Cr-labeled chicken, burro (BRC), and human red blood cells (HRC) in the presence of phytomitogens. PHA-stimulated cultures lysed all three types of targets, while PWM and Con A showed a "target cell specificity" for HRC and BRC, respectively. In mixtures of target cells only the appropriate targets were lysed by lymphocytes activated by either Con A or PWM indicating that soluble lymphotoxins do not play a major role in these reactions. Preincubation experiments suggested that there may be a population of pre-existing aggressor cells which only require linking to the targets by the mitogens for activation of their cytotoxic potential. Strong cytotoxic reactions were found with spleen cells, peripheral blood leucocytes, and bone marrow cells. Thymocytes were less active but could be stimulated for significant cytotoxicity, while bursal cells were generally unreactive. Spleen cells from agammaglobulinemic chickens totally lacking serum immunoglobulins and B cells with surface-bound immunoglobulins were as active as cells from normal chickens. The activity of spleen cells, from which phagocytic cells were removed was also unimpaired. These results indicate that the development of cytotoxic effector lymphocytes in mitogen-treated leucocyte cultures is a property of T lymphocytes. Although bone marrow cells fail to proliferate in response to these phytomitogens, they do have strong cytotoxic reactivity suggesting that different subsets of thymic-derived lymphocytes are responsible for mitogen-induced transformation and mitogen-induced cytotoxicity.

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.

Lymphocytes from bursa of Fabricius and thymus of chickens were purified and separated into the three cell subsets--T, B, and null cells--by the techniques of nylon fiber columns and cytotoxicity tests. The in vitro susceptibility of the fractionated lymphocytes to a virulent strain of infectious bursal disease virus (IBDV) was studied by using immunofluorescence as the infection criterion. B cells were highly susceptible. By contrast, T cells and null cells were insusceptible to infection by IBDV. The relationship between the target cells for virus infection and those B cells that possessed surface immunoglobulin (SIg) was tested. B cells were further divided into SIg(M)- and SIg(G)-bearing cells by immunoadsorbent columns employing anti-immunoglobulin M(IgM) (mu-specific) or anti-IgG (gamma-specific) sera coated with Sephadex. The SIg(M)-bearing cells were highly susceptible. These results suggest strongly that SIg(M)-bearing B cells were the target cells for infection by IBDV.

Lymphoid cells of the thymus and of the Fabricius bursa have been studied in 18-day-old chick embryos, normal and after injection of hydrocortisone on the 11th day of embryogenesis. By means of optical-structural computer analysis, the complex of morphometric and probability-statistic parameters of the nuclei in the lymphocytes are estimated: area of the nuclei, optical density of chromatin, asymmetry coefficient and variance. Normal T-lymphocytes possess less density of the nuclei, greater optical density of chromatin, greater values of negative asymmetry. The complex of these parameters can be used for identification of visually similar lymphoid cells of T- and B-lines. Under hydrocortisone effect structural changes of the nuclei in the thymus and Fabricius bursa lymphocytes of the chick embryo are uniform: increase in the area of the nuclei, decrease in optical density of chromatin, the asymmetry coefficient becomes positive.

B cell development in the bursa of Fabricius of the chicken was examined. We constructed neonatal bursa cell chimeras (F1 leads to parent, parent leads to F1) and studied the in vivo interaction of these chimeric B cells with host-derived T cells in adoptive cell transfer to determine whether there exists any environmental effect on B cells for MHC-restricted T-B cell interaction. The results indicate that F1 B cells that have developed in a parental host bursa still behave as normal F1 B cells and do not show any change in their MHC-restriction pattern. In addition, parent leads to F1 chimeric B cells were indistinguishable from normal parental B cells. B cells from all constructed chimeras, including fully allogeneic, responded well to the T-independent antigen Brucella. We conclude that the genotype of the B cell, and not the developmental environment, determines the MHC restriction phenotype of mature B cells.

The activities of 8 S and 6 S DNA ligases have been studied in the chicken lymphoid cells of blood, spleen and bursa of Fabricius at different stages of development, from late embryonic life to about 3 months after hatching. These cells have been sorted with the fluorescence-activated cell sorter FACS II on the basis of size and T or B antigenicity (immunofluorescence). The light 6 S DNA ligase has been previously demonstrated to be associated to a late stage of differentiation of thymocytes. In the bursa, a unique form of 8 S DNA ligase is found during the whole period of observation. This form of enzyme remains in the B cells of the spleen until 3 weeks after hatching, but is never present in the blood B cells. As far as T cells are concerned, the light DNA ligase is present in the blood from 18-day embryonic life on. In the spleen T cells, on the contrary, this enzyme appears only 3 weeks after hatching. Before this stage, splenic T cells are devoid of any form of DNA ligase activity. These findings show biochemical differences in T and B lymphocytes colonizing the periphery, blood and spleen, and suggest, at least for the T cells at early stages, a heterogeneity in the degree of differentiation.

Appropriately absorbed turkey antisera to antigenic surface determinants of chicken bursa (ABS) or thymus cells (ATS) were assessed for their selective immunosuppressive activy in vitro and in vivo. The intraperitoneal injection of ABS or ATS into 2-3-week-old normal white Leghorn chickens entailed a significant depletion of B or T cells respectively from spleen and peripheral blood, while bursa and thymus themselves remained unaffected. The potential of this 'peripheral serological bursectomy and thymectomy' paralleled that found after the conventional surgical procedures with subsequent sublethal irradiation. The mean survival time of skin allografts from donors of genotype B4B4 onto B8B8 recipients was significantly prolonged by treatment with ATS (29 plus or minus 12 days) as compared to untreated (14 plus or minus 2 days), normal turkey serum (12 plus or minus 3 days) or ABS-injected (13 plus or minus 2 days) groups. This selective suppression of a T cell-dependent immune reaction by ATS was also confirmed in vitro by its inhibitory action on the graft-versus-host reactivity of adult peripheral blood lymphocytes in the chorioallantoic membrane assay, where normal turkey serum and ABS were again ineffective. Thus, ABS or ATS produced in avian species may serve not only to delineate B and T cells in vitro, but can also be used for selective manipulation of immune reactions of the chicken in vivo.