Peptide and amine producing endocrine-like cells in the chicken thymus

Summary The thymus of the chicken contains at least two types of endocrine-like cells predominating in the juxtacortical medulla. One type stores 5-hydroxytryptamine and is stained by the argentaffin, chromaffin and Schmorl methods. Treatment with reserpine markedly reduces its 5-hydroxytryptamine content. The other cell type is devoid of 5-hydroxytryptamine; if supplied withl-dopa it can produce and store dopamine. Both cell types stain with the argyrophil method of Grimelius and with HCl-basic dye methods believed to reflect the presence of peptides with masked carboxyl groups. In the electron microscope both cell types were found to contain numerous cytoplasmic 2000–3000 ? granules similar to those seen in polypeptide hormone-producing cells elsewhere. The cytoplasmic granules in one of the two endocrine-like cell types are argentaffin and chromaffin, indicating that they are the storage site for 5-hydroxytryptamine. It is suggested that the main secretory products of the two endocrine-like cell types are peptides, possibly regulating lymphatic tissue function.     

Epithelial heterogeneity in the murine thymus: fucose-specific lectins bind medullary epithelial cells

By means of histochemical techniques, two lectins with nominal specificity for L-fucose, Tetragonolobus purpureas agglutinin (TPA), and Ulex europeus agglutinin (UEA) were found to specifically label the medullary area of murine thymuses. Although the binding of both lectins was restricted to the medullary area of the thymus, each staining pattern was unique. Cells binding UEA formed a reticular network throughout the medulla, whereas cells binding TPA occurred as single cells or small clumps of cells and resembled Hassall's corpuscles. The cells binding either lectin were identified as epithelial on the basis of ultrastructural features (tonofilaments, desmosomes, and keratohyalin bodies) and resembled Ia+ medullary epithelial cells described previously. An age-related decline in UEA binding was observed, whereas labeling with TPA remained unchanged. On the basis of the labeling patterns obtained with UEA and TPA and the reported specificities of these two lectins, it is suggested that the majority of the fucose detected is associated with type 1 carbohydrate chains.     

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.     

Cellular heterogeneity: yeast-side story

A major challenge for cells lies in their ability to detect, respond and adapt to changing environments that may threaten their survival. Among the numerous evolutionary strategies, cell-to-cell heterogeneity allows the emergence of different phenotypes within a population. This variability in cellular behaviors can be essential for a small fraction of cells to adapt and survive in various environments. Analyses at the single-cell level have allowed to highlight the great variability that is present between cells within an isogenic population. Numerous molecular mechanisms have been uncovered, allowing to understand the emergence and the role of cellular heterogeneity. These attempts at identifying the source of cellular noise have also provided clues for strategies needed to control heterogeneity. In this review, S. cerevisiae is used as an example to illustrate the different factors leading to cell heterogeneity, ranging from intracellular processes to environmental constraints. In addition, some recent strategies developed to modulate cell-to-cell variability are discussed.     

Cellular basis of graft-versus-host reactions

The biologic basis of Graft-Versus-Host Disease (GVHD) is presented as an extremely complex immunopathologic syndrome that involves interaction between many different donor and host cell types. A model of acute lethal GVHD was employed where adult unirradiated (DA X LEW)F1 rats were injected with LEW spleen and lymph node cells. Controls received the same dose of syngeneic cells. At intervals from 2 to 21 days after cell injection, GVHD and control animals were killed and nonadherent cell suspensions prepared from their lymph nodes, spleen and peripheral blood. Cell suspensions were treated with LEW-anti-DA-alloantiserum or normal LEW serum and then analyzed for sIgM+ (B cells), W 3/13+ (T cells), and IgG-Fc receptors (FcR). Evidence is discussed for the selective removal of host cells with the alloantiserum. In addition, the level of naturally cytolytic (NK/NC) cells was assessed by adding GVHD and control nonadherent lymphoid cells to heterologous lymphoma and sarcoma target cells. Evidence is presented that during acute GVHD, in this parental----F1 combination, there is an early increase within most compartments of donor as well as host W 3/13+ and W 3/13+FcR+ cells. NK/NC cells are increased as well at day 7. During middle stages of acute GVHD, host sIgM+ cells predominate. Late-stage acute GVHD rats contain few donor and host W 3/13+, W 3/13+FcR+, and NK/NC cells but many null cells most of which are FcR-. The importance of unraveling the nature of donor- and host-cell interactions occurring during acute GVHD, which result in rats whose lymphoid tissues are severely depleted of all nonadherent lymphoid cells but FcR- null cells, is discussed.     

Migration of bone marrow cells toward the thymus in C57BL/Ka under fractionated irradiation

Fractionated whole body X-irradiation (4 X 1.75 Gy at weekly intervals) induces a high percentage of thymic lymphomas in C57BL/Ka mice. These tumors develop after a long latency period during which the thymic lymphopoiesis is deeply altered. In the present work, we test wether those modifications are due to lack of prothymocyte homing to preleukemic thymuses. Our results show that the preleukemic state of the thymus don't prevent the homing of normal marrow precursors grafted immediately after an irradiation of 4 Gy. Thus the alterations of thymic lymphopoiesis observed after a leukemogenic irradiation are not due to a modification in the thymus receptivity to thymocyte precursors.     

Immunoreactive neurotensin and somatostatin in the chicken thymus

Summary Two distinct populations of endocrine cells in the chicken thymus display neurotensin and somatostatin immunoreactivity, respectively. Both cell types are few in number at hatching but proliferate rapidly during the first week. The neurotensin cells are Grimelius-positive and Hellerström-Hellmannegative. The somatostatin cells are Grimelius-negative and Hellerström-Hellman-positive. Both cell populations are non-argentaffin. The somatostatin-like material extracted from chicken thymus behaves immunochemically and chromatographically similar to synthetovine somatostatin, while the neurotensin-like material, from the thymus as well as from the gut, differs from synthetic bovine neurotensin in that it appears larger in size and more basic.     

Ontogeny of T cell receptors in the chicken thymus

A panel of murine mAb against chicken TCR and associated molecules was used to study the ontogeny of T cells. The intrathymic maturation of the TCR-gamma delta, (TCR-1) and TCR-alpha beta (TCR-2) sublineages was the focus of these studies employing immunoperoxidase staining of tissue sections and immunofluorescence analysis of cell suspensions. The first CD3+ cells appeared in the thymus on embryonic day 9 (E9) when the CD3 Ag was restricted to the cytoplasm. In tissue sections, both TCR-1+ and TCR-2+ cells were observed on E12, whereas only the TCR-1 cells were identifiable by surface immunofluorescence. On the next day, when a discrete thymic medullary region was first recognizable, the TCR-1 cells were present in both cortex and medulla. Two days later (E15), TCR-1 cells were found in the spleen. Surface TCR-2+ cells did not appear until E14, began to migrate in to the medulla on E17, and appeared in the spleen on E19. The first TCR-1 cells thus move quickly through this maturational pathway, whereas TCR-2 cells undergo a prolonged developmental period in the cortex. While most TCR-1+ cells were CD4-CD8-, a minor subpopulation (5 to 15%) were CD4-CD8+, and less than 1% were CD4+CD8+. In contrast, immature TCR-2+ thymocytes in the cortex were predominantly CD4+CD8+, whereas cells expressing a higher density of the CD3/TCR-2 complex were either CD4+CD8- or CD4-CD8+ and were localized in the thymic medulla. In the medulla of the mature thymus, the TCR-1+ cells preferentially occupy the cortico-medullary junction and form small aggregates around vessels. TCR-2+ cells were less frequent in these areas of TCR-1 accumulation. The thymic ontogeny and, by implication, the selection of the receptor repertoire thus differs substantially for these two TCR isotypes.     

The presence of immunoregulatory cells in chicken thymus: function in B and T cell responses.

The presence of immunoregulatory cells in chicken thymus was studied by using several different systems. Chickens injected with large numbers of syngeneic thymocytes were tested for their ability to produce antibody to heterologous red cells. Similar chickens were studied for their ability to reject allogeneic skin grafts. In separate studies, mixtures of thymocytes with spleen cells or with peripheral blood leukocytes were assayed for their ability to respond to PHA or to produce a graft-vs-host reaction in embryonic chicks. These studies indicated that immunoregulatory cells exist in chicken thymus, which displays both helper and suppressor activity. The suppressor cells were more prevalent or more easily detectable in young birds and in chickens with intact bursas. The helper function of thymocytes was seen to better advantage with cells derived from older animals and from bursectomized donors.     

Protease activity in fractionated blood cells of the vanadium accumulating ascidian Phallusia mammillata

Proteolytic activity was studied in the fractionated blood cells of the vanadium accumulating ascidian P. mammillata by separating the cells before measuring their activity. Cells were separated to avoid vanadocyte breakdown and subsequent vanadium diffusion into the assay medium. Our study revealed activity in the morula cell extract that was obtained by sonication and Centricon concentration. After removing part of the extract for enzyme activity assay the remainder was kept at 0 degrees C; it was later found that much of the protein in this latter fraction formed a sediment whereas the protease remained in solution. The serine-protease substrate specificity of the enzyme was measured and the results are discussed in relation to serine protease involvement in immune defense.     

Developmental changes in the cellular composition of the chicken thymus

The cellular composition of the chicken thymus has been analyzed at different ages by using size distribution analysis in combination with preparative cell electrophoresis. The combination of these two physical methods was able to clearly resolve two major cellular subpopulations in the young chicken thymus and suggested the exsistence of a third one. Microscopically, all three cell types appeared to be small lymphocytes. Medium and large lymphocytes are not detected as distinct peaks by the settings used.The analysis revealed dramatic developmental changes in the cellular composition of the thymus. The adult chicken thymus, which is known to have practically no cortex, contained mainly one relatively large cell type. This cell type may, therefore, represent the medullary lymphocyte and may be active in graft-versus-host (G.v.H.) reactions. In the early postnatal thymus that is known to contain little graft-versus-host reactivity this larger cell type was not detectable. Instead, smaller cell types were found to be dominant. The developmental shift from smaller to larger cells was discontinuous. Before thymus involution at 16 weeks of age, smaller and larger cells were both found to be present and to have the same typical size and electrophoretic mobility that is characteristic for the postnatal or the adult chicken thymus, respectively. Size and electrophoretic mobility were therefore taken as markers indicating distinct cellular subpopulation in the thymus.     

Analysis of the heterogeneity of chromatin proteins fractionated on hydroxyapatite

The protein composition of the liver chromatin has been studied by two techniques for fractionation of histones. The "lability" fraction of histones H2A-H2B is revealed. In these fractions histones H2B have many modified forms and they are not included into octamer (H3, H4, H2A, H2B)2. Young animals rather than old ones have much quantitative subfractions of histone H2B. The "lability" fraction of histones H2A-H2B is stated to be very significant in the activated and repressed chromatin structure.     

Analysis of cellular heterogeneity in mouse thymus cultures

Summary Analysis of 5 to 6 d primary cultures of cells derived from murine thymus glands revealed a heterogeneous population of cells rather than “pure” reticuloepithelial cell cultures as was assumed previously by other investigators. The monolayer cultures consisted of at least three cell types: thymus epithelial cells, macrophagelike epithelioid cells, and fibroblasts. Surprisingly, about 50% of the cells had positive cytochemical staining reactions for acid phosphatase and nonspecific esterase. The same cells phagocytized carbon particles, latex beads, and yeast. Furthermore, these cells could be removed from the initial cell suspension by phagocytosis of carbonyl iron, followed by magnetic separation, but once they had adhered to the substratum they were resistant to trypsin removal. All of these findings supported the conclusion that about 50% of the cells in the monolayers were macrophages. The other cells present were thymus epithelial cells and a small number of fibroblasts. Both of the latter types of cell were cytochemically negative, did not phagocytize particulate material, and were not removed by carbonyl iron treatment, but were removed by treating the monolayer with trypsin. The findings in this report indicated that epithelioid morphology alone was inadequate to identify correctly the cell types found in thymus cultures and that the use of such cultures as a model to study in vitro the maturation of certain immunological functions has been based on assumptions here shown to be incorrect. This work was supported by the Graduate School, The Ohio State University, the Bremer Foundation, the American Cancer Society (IN-16R), and National Cancer Institute Grant 5 ROL CA-19346-03.     

Cellular aging leads to functional heterogeneity of hematopoietic stem cells: a modeling perspective

Hematopoietic stem cells (HSCs) are the source for the life-long supply of functional cells in peripheral blood while they simultaneously maintain their own reserve pool. However, there is accumulating evidence that HSCs are themselves subject to quantitative and qualitative exhaustion. Although several processes linked to mitotic activity can potentially account for the observed aging phenomena (e.g., DNA damage, telomere shortening, epigenetic modification), a precise understanding of HSC exhaustion is still missing. It is particularly unclear how individual aging processes on the single-cell level translate on the phenotypic level of the overall tissue and whether there is a functional implication of an age-structured HSC population. We address these issues by applying a novel mathematical model of HSC organization in which division-specific, cumulative alterations of stem cell quality determine the phenotypic and functional appearance of the overall cell population. Adapting the model to a number of basic experimental findings, we quantify the level of additional heterogeneity that is introduced by a population of individually aging cells. Based on this model, we are able to conclude that division-dependent processes of cellular aging explain a wide range of phenomena on HSC exhaustion and that HSC aging needs to be considered as a highly heterogeneous process. We furthermore report that functional heterogeneity between young and old HSCs appears closely similar to the phenomena described for long- and short-term repopulating cells. We speculate whether differential, division-coupled stem cell aging introduces an intra-animal variability that also accounts for heterogeneity with respect to the repopulation ability of HSCs.     

Cellular heterogeneity in human epithelial neoplasms

Tumor cell heterogeneity has in recent years been the subject of numerous excellent review articles, but comprehensive reviews may not always distinguish between that which is known about tumors from direct observation and that which is inferred from the study of analagous systems. The purpose of this review is to describe what is known about cellular heterogeneity in human tumors and to discuss current models of the pathogenesis of cellular heterogeneity in light of the evidence available from the study of human cancer.