Suppression of the allograft response by implants of mature lymphoid tissues in larval Xenopus laevis

One hundred and twenty-two larvae of Xenopus laevis, the South African clawed toad, at developmental stages 48, 50, 52 and 54, were implanted in the tail with two allografts from adult tissues. In each case, one allograft was from kidney, while the other was either from kidney, thymus, spleen, or liver. In any particular host the two implants were always from the same donor and the implants were all visually matched in size. The experimental period was a maximum of nine days, so as to minimize the large numbers of changes normally accompanying larval progress from stage to stage. We are concerned with the timing of allograft response initiation under the implant conditions of each experimental group at a particular point in development. An allograft response was defined as an infiltration and accumulation of small lymphocytes in the “test” kidney allograft. Larvae of all stages developed allograft responses within one week post-implantation when the variable implant was from kidney, but implants from spleen and thymus suppressed both the timing of initiation and the subsequent intensity of the response. Spleen was more effective in this regard than thymus and both were more effective in the earlier larval stages. Liver proved to be toxic to the larvae. The relationship between the maturation of the lymphomyeloid tissues and external morphological staging is also discussed.     

Dynamics of apoptosis and proliferation in rat thymus and spleen during perinatal development (Ontogenesis)

The levels of spontaneous apoptosis and proliferation of the rat thymic and spleen cells, as well as their regulation by the hypothalamo-hypophysial system were studied during perinatal development. The apoptotic and proliferating cells in the thymus and spleen were assayed using flow cytometry with the DNA-specific dye propidium iodide. The level of apoptosis in the thymus reached 25% on day 18 of embryogenesis (E18) and decreased to 5% thereafter. In the spleen, the level of apoptosis gradually increased from 15 to 37% during the period of E18 to day 30 of postnatal development (P30). The level of proliferating cells in the thymus was 20–25% at all developmental stages studied. In the spleen, it was at a maximum on E18 (32%) and decreased almost twice on E21 (17%). On P7, the amount of proliferating cells again increased to 22% and then gradually decreased to 7% by P30. The surgical ablation of hypothalamus in utero on E18 did not affect cell apoptosis or proliferation in the thymus and spleen. The surgical ablation of both hypothalamus and pituitary led a twofold decrease of the level of apoptosis in the spleen and insignificant increase of the level of proliferation in the thymus. Thus, the numbers of cells in the embryonic thymus is regulated not only by the thymus itself, but also by the hypothalamo-hypophysial system. The programmed cell death in the embryonic spleen appears to be regulated by the hypothalamo-hypophysial system as well.     

Dynamics of apoptosis and proliferation in the rat thymus and spleen during perinatal development

The levels of spontaneous apoptosis and proliferation of the rat thymic and spleen cells, as well as their regulation by the hypothalamo-hypophysial system were studied during perinatal development. The apoptotic and proliferating cells in the thymus and spleen were assayed using flow cytometry with the DNA-specific dye propidium iodide. The level of apoptosis in the thymus reached 25% on day 18 of embryogenesis (E 18) and decreased to 5% thereafter. In the spleen, the level of apoptosis gradually increased from 15 to 37% during the period of E18 to day 30 of postnatal development (P30). The level of dividing cells in the thymus was 20-25% at all developmental stages studied. In the spleen, it was at a maximum on E18 (32%) and decreased almost twice on E21 (17%). On P7, the amount of proliferating cells again increased to 22% and then gradually decreased to 7% by P30. The surgical ablation of hypothalamus in utero on E18 did not affect cell apoptosis or proliferation in the thymus and spleen. The surgical ablation of both hypothalamus and pituitary led a twofold decrease of the level of apoptosis in the spleen and insignificant increase of the level of proliferation in the thymus. Thus, the numbers of cells in the embryonic thymus is regulated not only by the thymus itself, but also by the hypothalamo-hypophysial system. The programmed cell death in the embryonic spleen appears to be regulated by the hypothalamo-hypophysial system as well.     

Effect of splenectomy on the development of neonatal lymphoid organs transplanted to animals of varying age]

The effect of splenectomy on the development of newborn thymus and spleen grafted under the kidney capsule of young and old mice has been investigated. Preliminary splenectomy is shown to increase cell counts in grafted spleen that is more conspicuous in young recipients as compared with old ones. This result suggests a decrease with age in the inhibitory effect of the host spleen on the maturation of spleen grafted from newborn donor. Combined transplantation of newborn thymus and spleen has revealed a decrease of cell counts in the donor spleen grafted to the young splenectomized recipients and, on the contrary, increase of this parameter in old ones. Immune response in donor spleen with combined transplantation of the thymus to the old splenectomized recipients is much higher as compared with the same parameter in recipient without splenectomy. It is concluded that partial destruction of the old immune system is essential for its correction.     

Thymus-dependent and thymus-independent developmental pathways for peripheral T cell receptor-gamma delta-bearing lymphocytes

To elucidate the developmental pathways of T cells that bear TCR gamma delta, we have analyzed the kinetics of expression and biochemical characteristics of gamma delta receptors in the thymus and spleen of normal and athymic (nude) mice, as well as nude mice engrafted with neonatal thymuses. TCR gamma delta-bearing thymocytes and splenocytes have a CD4-8- phenotype, and both populations express products of the C gamma 1 locus. TCR gamma delta-bearing cells develop in the thymus before their appearance in the spleen. Young nude mice have no detectable TCR gamma delta-bearing cells in their spleens. When young nude mice are given thymus grafts, TCR gamma delta-bearing cells of host origin first develop in the engrafted thymus, followed by their appearance in the spleen. In the absence of a thymus graft, the spleens of old nude mice eventually develop small numbers of TCR gamma delta + cells, as well as TCR alpha beta + cells. These results demonstrate that there is a major thymic-dependent pathway for TCR gamma delta expression, as well as a minor thymic-independent pathway seen in older nude mice. The development of TCR gamma delta + cells in the thymus before their appearance in the spleen, both in normal ontogeny as well as in the thymus-engrafted nude mouse model, suggests that thymic TCR gamma delta + cells are precursors of the thymus-dependent population of peripheral TCR gamma delta + cells.     

Absence of mitochondrial uncoupling protein 3: effect on thymus and spleen in the fed and fasted mice

Mitochondrial uncoupling protein 3 (UCP3) is constitutively expressed in mitochondria from thymus and spleen of mice, and confocal microscopy has been used to visualize UCP3 in situ in mouse thymocytes. UCP3 is present in mitochondria of thymus and spleen up to at least 16 weeks after birth, but levels decrease by a half in thymus and a fifth in spleen after three weeks, probably reflecting the suckling to weaning transition. UCP3 protein levels increase approximately 3-fold in thymus on starvation, but expression levels in spleen were unaffected by starvation. Lack of UCP3 had little effect on thymus mass or thymocyte number. However, lack of UCP3 affected spleen mass and splenocyte number (in the fasted state) and results in reduced CD4+ single positive cell numbers and reduced double negative cells in the thymus, but as a 2-fold increase in the proportion of CD4(+), CD8(+) and DP cells in spleen. Starvation attenuates these proportionate differences in the spleen. A lack of UCP3 had no apparent effect on basal oxygen consumption of thymocytes or splenocytes or on oxygen consumption due to mitochondrial proton leak. Splenocytes from UCP3 knock-out mice are also more resistant to apoptosis than those from wild-type mice. Overall we can conclude that UCP3 affects thymocyte and spleen cell profiles in the fed and fasted states.     

Responsiveness of Thymus Cells to Syngeneic and Allogeneic Lymphoid Cells

THE thymus is necessary for the normal development of cell-mediated immunity in mice as shown by the immunological defects after neonatal thymectomy¹. Thymus cells themselves can be stimulated by allogeneic lymphoid cells in mixed leucocyte reaction (MLR)² and become killer cells or cytotoxic lymphocytes after stimulation with allogeneic spleen cells in vitro (H. Wagner and M. Feldmann, unpublished work) and in vivo^3,4. This suggests that the thymus as well as peripheral lymphoid tissues contain T cells which can be stimulated by foreign histocompatibility antigen to divide and differentiate into the cytotoxic lymphocytes which mediate cellular immunity. There have been suggestions that thymus cells might be stimulated to divide by “self” antigen, as well as foreign cells: incorporation of ³H-thymidine above background levels has been found in cultures with syngeneic spleen and thymus cells of adult rats⁵, although the experiments do not determine whether thymus or spleen cells have been stimulated. In contrast to these experiments, Howe et al. reported that only thymus cells of neonatal CBA mice reacted to allogeneic and syngeneic spleen cells of adult animals in “one way” MLR cultures^6,7. Whether the reaction of neonatal thymus cells to syngeneic adult spleen cells is recognition of “self” antigens is uncertain, since spleens of adult mice could carry antigens which do not occur in neonatal animals and are therefore “unknown” for neonatal thymus cells. We demonstrate here that neonatal thymus cells do not react to 4-day-old CBA spleen cells, but adult thymus cells do react against both allogeneic and syngeneic adult spleen cells.     

Effect of stromal mechanocytes of hematopoietic organs on in vitro formation of antibody producing cells

Cultured rabbit fibroblasts of bone marrow, thymus and spleen origin were added in spleen cell cultures in which the primary antibody response to SRBC was induced. Bone marrow fibroblasts caused strong inhibition of the response; thymus fibroblasts stimulated antibody formation; spleen fibroblasts inhibited the response when added in large amounts otherwise they produced no effect. The stimulation of antibody forming cell response by thymus fibroblasts proved independent of whether fibroblasts were irradiated or not. Bone marrow fibroblasts exhibited suppressive effect on the response predominantly during initial stages of antibody induction. All the 3 types of fibroblasts did not influence cell viability in spleen cells cultures, and were much more effective on addition to cultures of A-deficient spleen cells as compared to full spleen cells.     

Enhanced suppression of blastogenic responses by weanling mouse lymphoid cells treated with tetrahydrocannabinol in vitro

The suppressive effects of delta 9-tetrahydrocannabinol (THC) on the proliferation of lymphocytes from the spleen, lymph node, and thymus of weanling animals vs adult animals to the T-cell mitogen PHA were examined. THC had a suppressive effect on thymus cells from animals of both younger and older mice. THC suppressed spleen and lymph node cells responses to phytohemagglutinin (PHA) more readily when the cells were obtained from young mice rather than older animals. Suppression by THC in the adult mice was greater in an organ containing fewer mature T lymphocytes such as the thymus in comparison to lymphocytes in secondary organs such as the spleen and lymph nodes which contain more mature lymphocytes.     

Gangliosides as markers of cortisone-sensitive and cortisone-resistant rabbit thymocytes: characterization of thymus-specific gangliosides and preferential changes of particular gangliosides in the thymus of cortisone-treated rabbits

Neutral glycosphingolipids and gangliosides in rabbit thymus, spleen, bone marrow, and erythrocyte ghosts were analyzed by conventional chemical and enzymatic procedures and negative ion fast atom bombardment mass spectrometry (FABMS). Thymus gangliosides showed a characteristic composition. Major gangliosides comprising 75% of the total thymus gangliosides were sialosyl lacto-N-neo-tetraosyl- and sialosyl lacto-N-nor-hexaosylceramides containing NeuGc and palmitic acid. These major thymus gangliosides were not detected in spleen, bone marrow, or erythrocytes, whereas GD1a, which was not present in the thymus even in a trace amount, was present in spleen and bone marrow. In addition, the major gangliosides in rabbit thymus were preferentially reduced when an animal was given an intraperitoneal injection of cortisone acetate, as found on analysis 48 h later. The decrease was accompanied by a concomitant increase in NeuAc-containing GM3 with longer chain fatty acids.     

Starvation-sensitive UCP 3 protein expression in thymus and spleen mitochondria

To date, UCP 3 has only been associated with skeletal muscle and brown adipose tissue (BAT). Using RT-PCR/PCR methodology, we show that human spleen and human thymus contain UCP 3. In addition, using peptide antibodies, previously demonstrated to be selective for UCP 3, we show that UCP 3 protein is present in mitochondria isolated from rat thymus and mitochondria isolated from reticulocytes, monocytes and lymphocytes of rat spleen. UCP 3 protein expression is also starvation-sensitive. UCP 3 abundance is augmented in mitochondria isolated from thymus and mitochondria isolated from lymphocytes of the spleen from fasted rats when compared to fed controls. The results are consistent with a role for UCP 3 in developing lymphocytes, thymus atrophy and fatty acid utilisation in spleen and thymus.     

Cellular distribution of prothymosin alpha and parathymosin in rat thymus and spleen

By means of immunohistochemical methods, we have investigated the cellular distribution of prothymosin alpha and parathymosin in rat thymus and spleen, using specific antibodies raised against thymosin alpha-1 and against parathymosin. We observed prothymosin alpha immunoreactivity in lymphoid cells both in thymus and spleen. In the thymus, prothymosin alpha staining was more marked in cortex than in medulla. In the spleen, prothymosin alpha was found in lymphocytes of the periarteriolar lymphatic sheaths and was especially prominent in the germinal centers. Parathymosin immunoreactivity in the thymus was mainly localized in the medulla; positive cells were reticuloepithelial cells from the thymic reticulum and the blood barrier. Thymocytes were negative. In spleen, parathymosin was found in reticular cells arranged in a ring between the periarteriolar lymphatic sheath and the marginal zone. Our results do not support an exclusive role for these peptides as immune system hormones or cytokines.     

Effect of thymus polypeptide fractions on the development of rat thymus and spleen in organ culture

Peptides of the thymus--vilon, thymogen and thymalin, alone or in combination with concanavalin A, were used to investigate their effect on organotypic culture of thymus and spleen explants from 1- and 21-day old rats. Vilon, thymogen and thymalin in concentrations of 2 and 10 ng/ml and 5 ng/ml, resp., exerted stimulating effects in thymus and spleen tissue cultures from 21-day old rats as compared to the control explants. Vilon and thymogen showed inhibiting effect in the thymus tissue cultures from 1-day old rats as compared to the control explants. However, the peptides together with concanavalin A in concentration of 10 mkg/ml resulted in decreasing the action of concanavalin A alone. The polypeptide fractions of thymus and their synthetic analogs play different roles in the regulation of thymus and spleen development in rats of different age.     

Ontogeny of cell-mediated cytotoxicity: induction of CTL in early postnatal thymocytes.

In this study we characterized the time when cytotoxic T lymphocytes (CTL) can be induced in the thymus and spleen from their immediate CTL precursors (CTL-P). In contrast to fetal or newborn thymus, the thymus of 1 to 2-day-old C57BL/6 mice contained cells that, after cultivation in vitro with allogeneic DBA/2 stimulating cells, exhibited high levels (as great or greater than that induced in adult thymocytes) of CTL activity as measured by the ability to lyse P815 (DBA/2) tumor target cells. However, CTL activity induced in spleen cells remained how during the first 5 days of life, increased sharply between 6 to 9 days, and reached adult levels at 11 to 20 days. Furthermore, early postnatal spleen cells did not suppress the adult splenic CTL response. These results suggest 1) that the full potential to generate CTL in response to an allogeneic stimulus commences in the thymus on the first day after birth and 2) a different temporal appearance of immediate CTL precursors in the thymus and spleen.     

Calf thymus deoxyuridine triphosphatase differs from rat spleen enzyme in molecular disposition

Antiserum against calf thymus dUTPase was raised in rats by injection of the partially purified enzyme. The antiserum did not react with dUTPase purified from rat spleen, while antibody against rat spleen dUTPase partially reacted with calf thymus enzyme. Native molecular weight of the calf thymus dUTPase was estimated at 46,000 daltons by gel filtration, and the denatured form of the enzyme was about 22,000, as judged by immunoblot analyses using the antibodies. These results indicate that the calf thymus dUTPase is composed of two identical subunits.     

Studies on the regulation of lymphocyte production in the murine thymus and some effects of a crude thymus extract.

Cell proliferation in the murine thymus was studied in vivo under normal conditions and from 0 to 24 hr after a single injection of a water-soluble extract from mouse thymus, mouse spleen, and mouse skin. The thymus extract reduced during the first 24 hr the mitiotic activity 40%; the spleen extract had a weaker inhibitory effect. The skin extract had no such effect. The thymus extract and spleen extract inhibited the flux of cells into the S phase 0-8 hr after the injection of the extract. Initial labelling index was also reduced in this period. Eight hours after injection of the thymus or spleen extracts the inhibited cells initiated DNA synthesis. The rate of progression of blast cells through the cell cycle was normal 24 hr after the injection of the extracts. It was deduced from the analysis that the thymus extract inhibits processes triggering G0/G1 cells into DNA synthesis, the inhibition of G2 efflux being of minor importance. Finally a model for the regulation of proliferating thymic blast cells and the emigration of small lymphocytes from the thymus is proposed.     

Effect of pentoxyl, ibuprofen and acetylsalicylic acid on the thymus, spleen and adrenals in an experiment]

The effect of acetylsalicylic acid, ibuprofen and pentoxyl on the histological and morphometric pattern of the thymus and the weight of the thymus and spleen was studied in rats. There was decreased function of the thymus and its atrophy with acetylsalicylic acid and ibuprofen. Pentoxyl increased the secretory activity of the thymus. The effect of the drugs on the thymus and spleen was unidirectional.     

The role of the thymus in the establishment of suppressor cells in liver chimeras.

The role of the thymus in the establishment of specific suppression was studied in an experimental model in which lethally irradiated F1 mice were reconstituted with parental C57BL neonatal liver cells and then challenged with immunocompetent spleen cells syngeneic to the donor. In this model, inhibition of a graft-versus-host response by these spleen cells served as an indication of the establishment of a suppressive state towards syngeneic antigens in the liver chimeras. It was demonstrated that since thymectomy of the chimeras could not prevent the elicitation of a graft-versus-host response by spleen cells, the thymus is essential for the establishment of this specific suppression. Reconstitution of such chimeras with intact thymus grafts of either donor (C57BL) or host (F1) origin led to the inhibition of the graft-versus-host response and to the reappearance of the suppressive state. Removal of the thymus in intact liver chimeras after establishment of a suppressive state did not affect suppression. Thus, it was concluded that the thymus is needed in the chimeras during a critical period in the development of suppression. Once suppression is established, the presence of the thymus is no longer required.     

Effect of hypokinesia on nucleic acid and protein metabolism in the lymphoid organs of rats

Metabolism of nucleic acids and protein by lymphoid cells of the rat spleen and thymus was studied under conditions of 22-day hypokinesia. It was shown that in the course of hypokinesia the loss of cellular mass by the spleen and thymus was associated with varied biochemical changes in the remaining lymphoid cells. The thymocytes showed a significant activation of nucleic acid and protein biosynthesis. Meanwhile in spleen lymphocytes, DNA and RNA metabolism was inhibited with no appreciable changes in protein metabolism. Potential mechanisms of changes in metabolism of thymus and spleen lymphocytes under long-term hypokinesia are discussed.     

STUDIES ON THE REGULATION OF LYMPHOCYTE PRODUCTION IN THE MURINE THYMUS AND SOME EFFECTS OF A CRUDE THYMUS EXTRACT

Cell proliferation in the murine thymus was studied in vivo under normal conditions and from 0 to 24 hr after a single injection of a water-soluble extract from mouse thymus, mouse spleen, and mouse skin. The thymus extract reduced during the first 24 hr the mitotic activity 40%; the spleen extract had a weaker inhibitory effect. The skin extract had no such effect. The thymus extract and spleen extract inhibited the flux of cells into the S phase 0–8 hr after the injection of the extract. Initial labelling index was also reduced in this period. Eight hours after injection of the thymus or spleen extracts the inhibited cells initiated DNA synthesis. The rate of progression of blast cells through the cell cycle was normal 24 hr after the injection of the extracts. It was deduced from the analysis that the thymus extract inhibits processes triggering Go/Gi cells into DNA synthesis, the inhibition of G₂ efflux being of minor importance. Finally a model for the regulation of proliferating thymic blast cells and the emigration of small lymphocytes from the thymus is proposed.