Rat Thymus Cells Research Articles

Differences in the subcellular localization of the stimulatory effects of glucose and pyruvate on protein synthesis in rat thymus cells in vitro.

AbstractThymocytes incubated as cell suspensions in vitro are known to be markedly dependent upon added glucose for maintenance of maximal rates of incorporation of radiolabelled amino acids into protein. This requirement is only partially satisfied by other added substrates, such as pyruvate. Evidence is presented that incorporation of amino acids into protein associated with the nuclear fraction isolated from these cells is more dependent upon added glucose than is labelling of protein found in the rest of the cell.The dependence of the labelling of nuclear protein upon glucose is shown by comparing the ability of glucose and pyruvate to stimulate the incorporation of [14C‐L] valine into the protein of nuclear and cytoplasmic fractions of thymus cells. The fractions are isolated on sucrose gradients after incubating suspensions of cells in substrate‐free medium for two hours, adding carbohydrates and labelled L‐valine for 30 min and then stopping the incubation by breaking the cells with hypotonic shock. When the protein‐synthetic stimulatory effects of glucose and pyruvate are compared, glucose is almost equally capable (90%) at stimulating rates of protein synthesis in nuclear compared to cytoplasmic fractions. Pyruvate is much less effective in nuclear than in cytoplasmic fractions (30%).Evidence is also presented from pulse‐chase experiments that the glucosedependent labelling of protein associated with the nuclear fraction occurs within that fraction, as opposed to migration to the nuclear fraction after being synthesized elsewhere.It is suggested from these and other data that a unique ability of glucose to provide non‐mitochondrial ATP to the nucleus may be central to the dependence of the labelling of nuclear protein on this substrate.

Is cortisol metabolized as it dissociates from glucocorticoid receptors in thymus cells?

To investigate the possibility that metabolic transformations of a steroid hormone are associated with its release from nuclear receptors, rat thymus cells were incubated with [ 3H]-cortisol at 37°C to form nuclear glucocorticoid-receptor complexes. Then, by means of successive timed dilutions with buffer at 37°C, the receptor-bound radioactivity was isolated after it had been allowed to dissociate from the receptors and leave the intact cells under physiological conditions. Analysis of this radioactivity showed that it consisted of essentially pure [ 3H]-cortisol. It is therefore concluded that no metabolic transformation is necessary for or accompanies the release of Cortisol from the nuclear glucocorticoid receptors of thymus cells.

Isotope-dilution analysis of rate-limiting steps and pools affecting the incorporation of thymidine and deoxycytidine into cultured thymus cells.

1. Rat thymus cells were incubated in homologous serum (10%) and medium 199. The effects of various quantities of thymidine or deoxycytidine (0-30mum) on the radioactive labelling of cells with the corresponding radioactive deoxynucleoside were examined. From plots of the reciprocal of the radioactivity incorporated against the total deoxynucleoside concentration (;isotope-dilution plots'), values were obtained for (a) the V(max.) of the rate-limiting step governing incorporation of the deoxynucleoside, and (b) the concentration of the pool of compounds competing with the radioactive deoxynucleoside at that rate-limiting step. From changes in these values under different experimental conditions inferences were drawn on the position and control of the rate-limiting step within intact cells. 2. Isotope-dilution plots for deoxycytidine were linear, whereas plots for thymidine were bimodal, indicating an abrupt increase in both the V(max.) and pool concentration at a critical thymidine concentration (approx. 5mum). The bimodality was removed by amethopterin. The V(max.) determined with deoxy[U-(14)C]cytidine was approximately equal to the sum of the V(max.) determined with deoxy[5-(3)H]cytidine and the V(max.) determined with [Me-(3)H]thymidine at thymidine concentrations above 5mum. 3. The thymidine competitor pool at thymidine concentrations above 5mum was approximately equal to the sum of the deoxycytidine competitor pool and the thymidine competitor pool at thymidine concentrations below 5mum. The pools were independent of cell concentration and dependent on serum concentration. 4. These results were explained on the following basis. Deoxycytidine in serum (16mum) is the major source of both cytosine and, by way of thymidylate synthetase, thymine, in the DNA of thymus cells. Serum deoxycytidine normally maintains a sufficient intracellular concentration of dTTP to inhibit partially the activity of thymidine kinase. When the dTTP concentration is lowered, either by decreasing the concentration of deoxycytidine or by inhibiting thymidylate synthetase, the activity of thymidine kinase increases. The activity of thymidine kinase may also be increased by concentrations of thymidine greater than 5mum, which overcome the inhibition of the enzyme by dTTP. At concentrations of thymidine below 5mum, thymidine kinase limits the rate of labelling with [Me-(3)H]thymidine and the radioactivity is diluted by a pool of unlabelled thymidine in serum (4mum). At thymidine concentrations greater than 5mum, the activity of DNA polymerase limits the rate of labelling and the radioactivity is diluted both by serum thymidine and, indirectly, by serum deoxycytidine.

Suppression of cellular immunity in rats and mice by maternal treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin.

Treatment of female F-344 rats and C57B1/6 mice with 2,3,7,8-tetrachlorodibenzo-<i>p</i>-dioxin (TCDD) during the latter half of gestation and in the postnatal period resulted in a severe depletion of lymphocytes in the thymic cortex of the offspring. Cellular immunity was impaired in these animals. Allograft rejection times were prolonged in rats and mice. Graft-versus-host (GVH) activity of spleen cells as well as the response of rat thymus and spleen cells to phytohemagglutinin (PHA) was reduced on a cell-for-cell basis. No reduction occurred in the response of thymus cells to concanavalin A (Con A). Maternal treatment postnatally on day 0, 7 and 14 with 5 <i>μ</i>g TCDD/kg of body weight reduced total PHA and Con A response recoverable from the thymus by 91 and 74%, respectively. Histologically, depletion of thymic cortex was not due to lymphocyte destruction. There was no adrenal hypertrophy. Stress-induced release of glucocorticoids is not considered responsible for the immune suppression observed. In contrast to the marked effects in early life, reduced responsiveness of spleen cells to PHA in mice treated with TCDD when 1 month old was only seen at a dose level that was clearly toxic. In 4-month-old mice, PHA responsiveness as well as GVH activity were not decreased. Response of unstimulated and stimulated thymus and spleen cell cultures was not reduced by adding TCDD <i>in vitro</i>. In TCDD-exposed rats, decreased eosinophilia was observed in acidophilic cells in the adenohypophysis. This finding is discussed in view of the effect of TCDD on the thymus, and a possible mode of action of TCDD is proposed.

Lymphocyte Plasma Membranes

Abstract Plasma membranes prepared from rat thymocytes were used to immunize rabbits and after 6 months the resulting sera had cytotoxic titers of 1:1280 to 1:2560 for rat thymocytes. With the lactoperoxidase-catalyzed iodination reaction the thymus plasma membranes were labeled with 125Iodine, solubilized in non-ionic detergent, and immuno-precipitated with the cytotoxic rabbit anti-rat thymus sera. These sera bound 3.5 to 4.4% of the total acid insoluble counts while control sera bound 0.6%. Subsequently, the precipitates were solubilized, reduced, and characterized on SDS containing polyacrylamide gels. The labeled antigenic component proved to have a m.w. ∼45,000. The same antigenic material could be labeled by in vitro incubation of rat thymus cells in 14C amino acids. Absorption studies suggested that this antigenic membrane constituent was shared by all lymphoid cells and represented a major immunodominant area on lymphocyte surfaces during heterologous immunization.

Cell to cell potassium transport in a monolayer of malignant and normal rat thymus cells

Cell to cell potassium transport in a monolayer of malignant and normal rat thymus cells

Steroid-binding properties and stabilization of cytoplasmic glucocorticoid receptors from rat thymus cells.

1. A competitive binding assay was adapted for determination of the specific binding of glucocorticoids to cytoplasmic receptors from rat thymus cells. The steroid-receptor complexes prepared by incubation of a cytoplasmic fraction from rat thymus cells with [1,2-(3)H(2)]cortisol or with [1,2,4-(3)H(3)]triamcinolone acetonide had rates of dissociation at 37 degrees C similar to those from intact cells. 2. The cytoplasmic receptor was unstable at 3 degrees C, but the rate of inactivation was decreased in the presence of 2.5mm-EDTA. The steroid-receptor complex was stable. 3. Rate constants for association and for dissociation, and association constants, were determined for the interactions of cortisol, cortexolone, dexamethasone and triamcinolone acetonide with the cytoplasmic receptor at 3 degrees C. Differences in the association constants for different steroids could largely be accounted for by the differences in the rate constants for dissociation, but the rate constants for association did not vary greatly; the implications of these findings for the nature of the steroid-binding site are discussed. 4. A cytoplasmic fraction prepared from cells which had been incubated at 37 degrees C under anaerobic conditions bound much less [1,2-(3)H(2)]cortisol than did a fraction from aerobic cells, but the binding capacity was restored after exposure of the anaerobic cells to O(2). 5. The specific binding of [1,2-(3)H(2)]-cortisol to intact thymus cells incubated aerobically was not affected by the presence of 0.1mm-cycloheximide, nor did this concentration of cycloheximide inhibit the recovery of specific binding observed when anaerobic cells were transferred to an aerobic atmosphere. 6. The energy dependence of specific binding of cortisol to the receptor is discussed with reference to possible mechanisms.

Rabbit anti rat lymphocyte serum: immunosuppression mediated by a pure IgM fraction.

Eleven rabbits were immunized i.v. weekly for 3 weeks with rat thymus cells during azathioprine administration. The antisera were pooled and fractionated on Sephadex G‐200 to obtain IgM and IgG fractions. The lymphocytotoxic activity was stronger in the IgM than in the IgG fraction. Whole serum, the IgM fraction and the IgG fraction prolonged the survival time of skin allografts from 12.0 to 16.5, 19.2 and 14.4 days respectively. It is concluded that IgM antibodies in antilymphocyte sera also can exhibit immunosuppressive activity.

Time Course of Early Events in the Action of Glucocorticoids on Rat Thymus Cells in Vitro: SYNTHESIS AND TURNOVER OF A HYPOTHETICAL CORTISOL-INDUCED PROTEIN INHIBITOR OF GLUCOSE METABOLISM AND OF A PRESUMED RIBONUCLEIC ACID

Abstract Previous work has shown that physiological amounts of cortisol added to rat thymus cells in vitro begin to inhibit glucose transport (as measured by the accumulation of glucose 6-phosphate during a 5-min glucose pulse) by 15 to 20 min; the effect of cortisol is blocked by actinomycin D present from 0 to 5 min, suggesting a rapid stimulation by cortisol of RNA synthesis. It is now shown that during the period from 15 to 30 min, but not prior to 15 min, the hormone effect is blocked by cycloheximide or puromycin. The coincidence in time of sensitivity to these inhibitors of protein synthesis with the development of inhibition of glucose transport, together with our earlier results, has led us to postulate that the hormone effect is expressed through induction of a protein that rapidly inhibits glucose transport. The preceding RNA-synthetic step possibly represents synthesis of mRNA for this protein. The previously demonstrated temperature-sensitive step is shown to lie between the RNA- and protein-synthetic steps. It perhaps corresponds to transfer of mRNA from nucleus to cytoplasm. Decay rates of the hypothetical cortisol-induced RNA and protein have been measured following removal of hormone from specific binding sites or addition of inhibitors of RNA or protein synthesis. The protein appears to have a life span of not more than 2 hours at 37°, while the RNA has a life span of less than 40 min.

Corticosteroid binding macromolecules in the nucleus and cytosol of rat thymus cells

Rat thymus cells and cell free fractions were incubated with [ 3H]cortisol. Nuclear and cytosol cortisol-binding macromolecules could be detected by Sephadex gel filtration and sedimentation in sucrose gradients. The nuclear receptor binds [ 3H]cortisol directly. On the basis of equilibrium dialysis data a K s of 4.8 · 10 9 M −1 has been determined. The molecular weight of the nuclear receptor is approx. 150 000 (approx. 7 S) that of the cytosol binding protein approx. 70 000 (approx. 4 S). Co-chromatography on DEAE-cellulose of [ 3H]cortisol-labelled cytosol and [ 14C]cortisol-labelled rat serum proteins shows that the 4-S cytosol corticosteroid-binding protein has similar elution characteristics to serum transcortin.

Glucocorticoid-receptor complexes and the earliest steps in the action of glucocorticoids on thymus cells

Cortisol and other glucocorticoids added at physiological concentrations to rat thymus cells in vitro at 37°C begin to inhibit glucose transport after about 15 min. This effect corresponds to effects observed in vivo and probably is in good part responsible for the catabolic actions of glucocorticoids on lymphoid tissue. From a variety of experiments we have concluded that cortisol initially stimulates synthesis in the nucleus of a specific form of RNA that. after an intermediate step, by 15 min initiates synthesis of a protein that inhibits glucose transport. The first step in cortisol action is formation of specific cortisol receptor complexes. At 37°C this process is complete by 7 min, by which time the complexes are localized largely in the nucleus. At 3°C, however, most of the complex appears in the supernatant when cells are broken by osmotic shock and nuclei spun down. This non-nuclear bound complex we refer to as “cytoplasmic”. On warming the cells to 37°C, [ 3H]-cortisol bound in the cytoplasmic complex becomes bound in the nucleus within l min, probably by transfer of the cytoplasmic complex in loto to a nuclear acceptor site. Isolated cytoplasmic and nuclear receptors bind glucocorticoids specifically, becoming saturated over the physiological range of glucocorticoid concentrations. The isolated cytoplasmic receptor has a half-life at 3°C of about 2 h. Saturating concentrations of cortisol or other glucocorticoids increase this value to more than 20 h. Both α and β sides of the steroid appear to interact with the binding site. which probably consists of a hydrophobic pocket with polar groups that form hydrogen bonds. The principal driving forces for formation of the hormone-receptor complex are probably hydrophobic interactions, the hydrogen bonds conferring specificity to the interactions. Cortisol-eytoplasmic receptor complexes are transformed by brief warming at 25°C into complexes with high affinity for nuclei. These latter complexes become rapidly associated with isolated nuclei at 3°C. Two alternative functions are proposed for the hormone in this temperature-sensitive transformation: one is that hormone binding displaces the equilibrium of the receptor towards a form with high affinity for nuclei; the other assumes that the cytoplasmic receptor is in a non-equilibrium state, and that hormone binding accelerates its transformation to the equilibrium high-affinity form. Protein synthesis does not appear to bo necessary to replace receptors that are transferred to the nucleus in the presence of hormone. ATP or some related substance, however, does seem to be required, it is proposed that ATP generates the normal cytoplasmic receptor from a pre-cursor. Furthermore, it is suggested that the precursor may be the form in which the receptor leaves the nuclear site, so that in the presence of hormone there is a continuously operating receptor cycle, dependent for energy on ATP.

Limited synergism between rat thymus and mouse bone marrow.

IN a number of experimental systems1 it has been shown that thymus and bone marrow-derived cells can cooperate in the production of humoral antibodies. In some instances this synergism has been demonstrated when the interacting cells are antigenically similar but in other experiments allogeneic cells have been found to cooperate. We have now demonstrated cooperation between rat and mouse cells. A two-step experiment was performed, following the design of Miller2. Rat or mouse thymus cells were introduced with antigen into irradiated recipients. The donor cells were collected and injected into secondary host mice. The details of these experiments and their results are as follows.

RNA dependent DNA polymerase in C type particles from normal rat thymus cultures.

THE isolation and characterization of C type particles released from normal rat thymus cell cultures has been described1, 2. The detection of RNA dependent DNA polymerase activity in these C type particles is reported here. Some properties of the enzymatic activities found in C type particles isolated from normal rat thymus cultures are described and compared with DNA polymerase found in isolated Moloney leukaemia virus (MLV) particles purified from the growth media of rat thymus cultures infected chronically with MLV2. The sensitivity to actinomycin D is different in the two kinds of C type particles, suggesting that the DNA polymerases are not identical.

Incorporation of amino acids into proteins catalyzed by prostate nuclei

Purified rat ventral prostate nuclei, lacking the outer layer of their nuclear envelopes, incorporated radioactive amino acids into hot trichloroacetic acid-insoluble materials. Some properties of this reaction examined include: kinetics, pH and temperature dependence, effects of possible inhibitors, and of changes in concentration of nuclei or radioisotope. Association of radioactivity with incubated nuclei was demonstrated by light microscope autoradiography. Labelled proteins extracted from prostate nuclei and microsomes and examined by polyacrylamide gel electrophoresis yielded dissimilar patterns. This limited incorporation, catalyzed by prostate nuclei, resembles that described with rat thymus, liver and HeLa cell nuclei. While its biochemical role as reflected by assay in vitro is obscure, administration of androgen restored the reduced activity that followed castration; the process is partially androgen-dependent.

Evidence for Irreversible, Actinomycin D-sensitive, and Temperature-sensitive Steps following Binding of Cortisol to Glucocorticoid Receptors and Preceding Effects on Glucose Metabolism in Rat Thymus Cells

Following cortisol binding to the specific glucocorticoid receptors of thymus cells at 37° (a process which takes about 7 min), there is a 5- to 10-min lag period before a cortisol effect can be observed on glucose 6-phosphate accumulation after a glucose pulse. Evidence is presented that during this period the signal initiated by cortisol binding traverses an irreversible, an actinomycin D-sensitive, and a temperature-sensitive step. The irreversible step is shown by the fact that removal of cortisol from the glucocorticoid receptors (by displacement with cortexolone, a metabolically inactive glucocorticoid competitor, or by washing) well before any metabolic effect has appeared does not prevent the subsequent appearance of the cortisol effect. The actinomycin D-sensitive step is shown by the fact that, whereas addition of actinomycin D to thymus cells together with cortisol prevents the cortisol effect from developing subsequently, addition of actinomycin D 5 min after cortisol does not prevent the cortisol effect. To produce these actions, actinomycin D must be used at 100 µg per ml. This same concentration is necessary to inhibit RNA synthesis rapidly in thymus cells by about 80%. The temperature-sensitive step is shown by the fact that the duration of the lag period preceding the appearance of a cortisol effect increases markedly at temperatures below 37° to more than 120 min at 20°. The duration of the lag period at 37° can be shortened if cells are first incubated with cortisol at 20°. The temperature dependence of the irreversible step is such that it cannot be identical with, but must precede, the temperature-dependent step. The actinomycin D-sensitive step may or may not be identical with one of the other two steps.

Ontogeny of cellular immunity: Development in rat thymocytes of mixed lymphocyte reactivity to allogeneic and xenogeneic cells

The one-way mixed lymphocyte reaction of rat thymocytes to allogeneic cells was studied, using ACT and Fischer rat strains. Mitomycin-treated thymocytes or thymocytes from F 1-hybrid rats were used as the stimulating cells. The reactivity of neonatal rat thymus cells was somewhat lower than that of adult rat thymocytes, but was nevertheless quite significant. No reactivity could be determined in neonatal rat thymocytes to mitomycintreated xenogeneic (mouse) cells, although thymocytes from 7-day-old rats reacted equally well to allogeneic and xenogeneic cells. Graft-versus-host reactivity of both rat and mouse thymus cells was also determined. It was found that neonatal and adult rat thymocytes, as well as adult mouse thymocytes, could induce splenomegaly in neonatal mice, but not in neonatal rats. The implications of these findings are discussed in the light of recent theories linking reactivity to histocompatibility antigens to antibody formation.

Antibody-induced lysis of nucleated cells in agar gel.

Cytolysis of nucleated cells incorporated into agar gel was studied using hetero- and isoantibodies. Evidence was presented that IgG and IgM antibodies may participate in these reactions. Studies on the lytic activity of rabbit anti-rat sera showed that antibodies inducing lysis of rat thymus cells are directed against species-specific cell surface antigens. Evidence was presented that some of these antigens are shared by nucleated cells and erythrocytes whereas other antigens are present on nucleated cells only. Tests with rabbit anti-sheep erythrocyte serum demonstrated that Forssman antibodies elicit lysis of nucleated cells originating from Forssman-positive species. It was also shown that infectious mononucleosis sera are capable of inducing lysis of sheep and bovine nucleated cells. In studying human renal transplantation sera, cytolysis of kidney cells was induced by sera obtained from recipients after removal of rejected grafts or pretransplantation sera of recipients who rejected hyperacutely their grafts. These results were consistent with those observed in previous studies by means of mixed agglutination.

CELLULAR DIFFERENTIATION IN THE THYMUS

Young adult rat thymus and lymph node cell subpopulations were obtained by differential flotation on discontinuous BSA density gradients and assayed for properties characteristic of mature thymus-derived lymphocytes. One such subpopulation (C) of thymocytes was enriched in its ability to respond mitotically to a hemiallogeneic MLR stimulus, to localize in the parenchyma of lymph nodes and spleen, and to initiate a GVH reaction in a suitable host. These cells did not respond well to mitotic stimulation by PHA, they were lighter in density than the majority of mature lymph node thymus-derived lymphocytes, and they possessed a thymus-specific antigen (RTA) not present on peripheral lymphoid cells. We conclude that the acquisition of peripheral properties occurs sequentially, during an intrathymic differentiation cycle or shortly after the cells leave the thymus.

Alterations in the phosphorylation of (3H)uridine and RNA synthesis in rat thymus cells after glucose depletion and treatment with prednisolone.

Alterations in the phosphorylation of [3H]uridine and in RNA synthesis resulting from restrictions in the availability of glucose as the only source of energy were studied in rat thymus cells in vitro and compared to the influence of prednisolone injected in vivo on both parameters. The following results were obtained: 1 In the absence of glucose, an impairment in the phosphorylation of [3H]uridine is observed after 15–30 min of incubation. A corresponding decrease in the formation of RNA is not clearly established before 60 min of incubation. 2 Fluctuations in the concentration of glucose in the incubation medium between 1.0 mg and 0.1 mg of glucose/ml produce only modest changes in the capacity of rat thymus cells to synthesize RNA. 3 Phosphorylation of [3H]uridine and RNA synthesis appear to be inhibited in a synchronous rather than a sequential fashion after the administration of prednisolone. 4 The phosphorylation of [3H]uridine is also inhibited in rat thymus cells under the influence of prednisolone when RNA synthesis is completely blocked by actinomycin D or by incubation of thymus cells at 0°. 5 On the other hand, thymus nuclei from prednisolone-treated rats which were depleted of their capacity to phosphorylate [3H]uridine by repeated treatment with a buffer solution containing Triton X-100, still displayed an impairment in their capacity to utilize [3H]ATP for RNA synthesis as compared to corresponding nuclear preparations from normal animals. 6 The steroid-mediated decrease in the phosphorylation of [3H]uridine does not reflect a direct steroid-effect on the kinases but appears to be caused by changes in the structural environment to which these enzymes are normally bound. Our results appear to indicate that prednisolone exerts its catabolic or antianabolic effect on rat thymus cells by at least two distinct mechanisms, one of which involves a decrease in precursor phosphorylation while the other leads to an impairment of RNA synthesis.

COOPERATION OF LYMPHOID CELLS IN AN IN VITRO GRAFT REACTION SYSTEM

SUMMARY The activity of rat lymphocytes from different organs was compared in an in vitro system based on sensitizing rat lymphocytes on C3H mouse fibroblast monolayers and then testing the lytic effect of transformed lymphocytes (large pyroninophilic cells) against the C3H cells. Thymic cells were found to manifest very low lytic activity, spleen cells were significantly more active than thymus cells, and lymph node cells had the highest activity. These differences could not be attributed to differences in the extent of transformation, since thymus cells sensitized on C3H fibroblasts produced at least as many large pyroninophilic cells as did lymph node cells. We assumed that rat thymus cells which can respond by transformation to mouse monolayers may play a role in determining the lytic process of other lymphocytes. To test this hypothesis, thymus cells were added to spleen cells and the admixed cell population did indeed show an increased lytic effect, compared to thymus or spleen cells alone. Rat thymus cells, presensitized against C3H mouse fibroblasts (8-13 days in culture), were mixed with fresh spleen cells and the mixture was tested for lysis on C3H monolayers. An enhanced lytic activity was manifested, appearing earlier than when any other lymphoid cell combinations were tested in an identical manner. The antigenic specificity of this system was directed by the presensitized thymus cells, which were found to be capable of interacting effectively with nonsensitized spleen cells, even when cultured on syngeneic cells after sensitization and before being mixed with the spleen cells. We suggest that the in vitro graft reaction system is based on an interaction between lymphoid cells, in which thymus cells act as antigen-recognizing cells, and that thymus cells may participate in or fulfill the role of memory cells.