A novel early effect of concanavalin A on thymocytes.

Concanavalin A, added to freshly isolated rabbit thymocytes, markedly enhanced the extracellular appearance of non-immunoglobulin proteins. Time course studies revealed that the onset of enhancement occurred virtually without delay. The effect appeared to be restricted only to certain of the thymus-derived cells because thymocytes obtained from rabbits treated with hydrocortisone, as well as splenocytes derived from untreated rabbits essentially did not exhibit the enhancement. Stimulation by concanavalin A was specific in that pokeweed mitogen and lipopolysaccharide were without effect and also in that α-methyl-mannoside, but not galactose, abrogated the concanavalin A-mediated enhancement. Experiments with mouse thymocytes demonstrated that the cells which responded to concanavalin A were primarily cells that bear the θ-antigen on their surface (T-cells).     

Phosphoinositide metabolism and the calcium response to concanavalin A in S49 T-lymphoma cells. A comparison with thymocytes.

Comparisons were made between transformed S49 T-lymphoma cells and normal murine thymocytes in their polyphosphoinositides, inositol polyphosphates and cytosolic free calcium concentrations ([Ca2+]i), and the effects of the T-cell mitogen concanavalin A (Con A) on these properties. 1. The ratios of the polyphosphoinositides to phosphatidylinositol in both exponential-phase S49 cells and mitogen-stimulated thymocytes (G1 phase) were greater than in quiescent (G0-phase) thymocytes. 2. In response to Con A, the amount of phosphatidylinositol 4,5-bisphosphate (PtdInsP2) in S49 cells decreased slightly (17% in 30 min), and this was sufficient to account for the small amounts of inositol phosphates that accumulated. In contrast, it has been shown previously that Con A stimulates a rapid resynthesis of PtdInsP2 in thymocytes and the amounts of inositol phosphates released rapidly exceed the steady-state amount of the PtdInsP2 precursor [Taylor, Metcalfe, Hesketh, Smith & Moore (1984) Nature (London) 312, 462-465]. 3. The [Ca2+]i did not differ significantly in S49 cells and thymocytes before the addition of Con A, and the increases in [Ca2+]i in response to Con A were similar in both types of cell. 4. The [Ca2+]i increase in response to Con A was inhibited by similar concentrations of intracellular cyclic AMP (2-10 microM) in S49 cells and thymocytes, suggesting that similar regulatory mechanisms act on this response in both types of cell. The data demonstrate that the basal [Ca2+]i and phosphoinositide metabolism is similar in both the normal cells and their transformed counterparts. In addition, they suggest that the activated Con A receptors generate very similar signals in the two cell types, and that any perturbations of primary signal transduction to the secondary phosphoinositide and [Ca2+]i responses in the S49 phenotype are quantitative rather than qualitative.     

Phospholipase A2 inhibitory activity in thymocytes of dexamethasone-treated mice--possible implication of lipocortins

The cellular phospholipase A2 activity of mouse thymocytes was estimated in vitro by the release of [3H]-Arachidonic acid from labeled and calcium ionophore A23187-stimulated cells. This activity was decreased in thymocytes from dexamethasone-treated mice. Thus, the presence of phospholipase A2 inhibitory proteins in mouse thymus was investigated. Three main proteins (36 kDa I, 36 kDa II, 73 kDa) were purified. These proteins were able to inhibit both phospholipase A2 in vitro, and the release of [3H]-Arachidonic acid from labeled and stimulated mouse thymocytes. Biochemical analysis revealed that the three proteins were lipocortin-like proteins. Our results show that in vivo dexamethasone treatment induces a phospholipase A2 inhibitory activity in mouse thymus, such an inhibition can be reproduced on isolated thymocytes by purified thymic lipocortins, known as glucocorticosteroid-inducible proteins.     

Studies on phytohemagglutinins: XXII. Isolation and characterization of a lymphocyte receptor for concanavalin A

A receptor glycopeptide for concanavalin A was isolated from calf thymocytes by a method originally devised for the isolation of a lectin receptor from human erythrocytes (Kubánek, J., Entlicher, G.; and Kocourek, J. [1973] Biochim, Biophys. Acta 304, 93–102). The method consisted of pronase digestion of the lipid-depleted thymocyte membrane material followed by ethanol fractionation, separation on Sephadex and preparative paper electrophoresis. The isolated glycopeptide contains 10.4% of neutral sugar. The molar ratios of the sugar components mannose, galactose, glucosamine, glucose, fucose and sialic acid are 3 : 2 : 2 : 1 : 1 : 1. The minimum molecular weight calculated from the sugar composition is about 12 000.Concanavalin A receptor activity of the glycopeptide was demonstrated in three different ways: (i) Reduction of the ¹²⁵I-labeled concanavalin A binding to thymocytes. (ii) Prevention of concanavalin A induced agglutination of calf thymocytes. (iii) Inhibition of concanavalin A stimulated DNA synthesis in calf and rabbit thymocytes and rabbit lymph node lymphocytes cultivated in vitro.The isolated glycopeptide seems to be involved in the interaction of lymphocytes with concanavalin A and the subsequent stimulation.     

Effect of cyclosporin A on lymphopoiesis. II. Developmental defects of immature and mature thymocytes in fetal thymus organ cultures treated with cyclosporin A

The effect of cyclosporin A (CsA) on early T cell development was studied by two-color flow cytometric and biochemical analyses using the fetal thymus organ culture system. Addition of CsA to organ culture resulted in a decreased cell yield and complete inhibition of the appearance of TCR-alpha beta-bearing, single positive thymocytes (both CD4+CD8- and CD4-CD8+). Furthermore, the generation of CD4+CD8+ thymocytes was markedly inhibited by CsA treatment, whereas the development of CD3-, CD4-CD8+ thymocytes and TCR-gamma delta-bearing, CD4-CD8- thymocytes was not affected. These results suggest that CsA induces a maturational arrest of T cells entirely within the thymic environment, and indicate that CsA-induced inhibition occurs at more than one stage of intrathymic T cell development.     

The intercellular interactions in the interphase death of irradiated thymocytes. A study of the nature of the mediator of the interaction of irradiated thymocytes]

A study was made of the influence of cytosol and conditioned medium, obtained at different times of incubation of irradiated thymocytes, on native thymocytes, as well as of the influence of mixing the suspension and changing the medium and the enzyme inhibitors, involved in metabolism of arachidonic acid, on the radiation injury to thymocytes. Cytosol of thymocytes, incubated for 60 min after irradiation, was shown to exert an insignificant toxic effect on native thymocytes. The conditioned medium of irradiated thymocytes is virtually ineffective. The mixing of the suspension upon incubation and the changing of the medium reduced the damage to exposed thymocytes while inhibitors of phospholipase A2 and lipoxygenase prevented completely the radiation-induced pyknosis of nuclei, whereas a inhibitor of cyclooxygenase did not influence it. It is inferred from the data obtained that unstable lipoxygenase products may serve as mediators of intercellular interactions of exposed thymocytes.     

Unidirectional K+ fluxes in rat thymocytes stimulated by concanavalin A.

Unidirectional K+ fluxes were estimated in isolated rat thymocytes by 42K exchange kinetics. The cells were either preloaded with isotope and the release of it measured during incubation for one hour at 38 degrees C, or the cellular uptake of isotope during a similar incubation was measured. The influx rate of untreated thymocytes was: 2.3-10(-12) moles cm-2-s-1 and efflux rate: 1.8-10(-12) moles cm-2-s-1. When con A was added to the cells, influx was raised 74% and efflux 65%. Maximal effect was obtained when the concentration of con A was 15 mug/ml, but concentrations as low as 0.75 mug/ml were effective. Hydrocortisone resistant thymocytes responded at least was well as untreated cells to con A, which also raised RNA synthesis rate in the former cells 2.5 times. Using an extracellular marker, 51CrEDTA, intracellular concentrations of some ions was estimated in the thymocytes after one hour incubation: Na+: 30 mmoles/kg water, K+: 177 mmoles/kg water and Cl-:43 mmoles/kg water. Cellular water content: 69%. These values were not found significantly altered when con A was present. Since con A raised influx and efflux to the same extent and no net flux of K+ could be detected, it is proposed that both active and passive transport of K+ was increased by con A. The increased fluxes induced by con A, can apparently not be reversed by removal of con A from the incubation medium or by addition of the inhibiting hapten, alpha-methyl-D-mannoside.     

A cytocidal tissue kallikrein isolated from mouse submandibular glands

A cytocidal factor against mouse thymocytes was purified from the submandibular glands of female BALB/c mice using Sephadex G-50 gel filtration chromatography and reverse-phase HPLC. SDS-PAGE and amino acid sequence analysis revealed that the cytocidal factor was mouse glandular kallikrein (mGK)-6. mGK-6 showed an optimal enzyme activity at pH 10 and a cytocidal activity against thymocytes in a dose-dependent manner.     

Early plasma-membrane-potential changes during stimulation of lymphocytes by concanavalin A

1. We have monitored the plasma-membrane potential of lymphocytes by measuring the accumulation of the lipophilic cation methyltriphenylphosphonium (TPMP+) in the presence of the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP). 2. The mitogen concanavalin A causes a decrease in TPMP+ accumulation by pig lymphocytes corresponding to a 3 mV depolarization with 2 1/2 min. Concanavalin A does not alter 86Rb+ uptake in the first 30 min. 3. In contrast concanavalin A increased TPMP+ accumulation and the rate of Rb+ uptake in mouse thymocytes. This is consistent with a previous proposal that the mitogen induces a hyperpolarization of mouse thymocytes as a result of stimulation of a Ca2+-dependent K+ channel. 4. Studies with the calcium ionophore A23187 and quinine (an inhibitor of the Ca2+-dependent K+ channel) suggest that the channel is partially closed in mouse resting thymocytes but is almost fully active in pig resting cells. Thus concanavalin A hyperpolarizes mouse thymocytes by activating the Ca2+-dependent K+ channel but cannot do so in pig lymphocytes because the channel is already maximally activated. 5. The 3mV depolarization of pig cells cannot be explained by a decrease in electrogenic K+ permeability.     

Regulation by interleukin 2 of interleukin 2 receptors and gamma-interferon synthesis by human thymocytes: augmentation of interleukin 2 receptors by interleukin 2

The role of interleukin 2 (IL 2) on the expression of IL 2 receptors and on the synthesis of gamma-interferon (gamma-IFN) by human thymocytes was investigated. Human thymocytes isolated from specimens obtained during cardiac surgery of infants and children were induced with one or all of the following agents: IL 2, concanavalin A (Con A), and 12-O-tetradecanoylphorbol 13-acetate (TPA). The expression of IL 2 receptors and gamma-IFN titers were determined. The results indicate that thymocytes cultured in complete medium do not express receptors for IL 2, nor did IL 2 by itself induce the expression of IL 2 receptors. Con A induced the expression of IL 2 receptors by a moderate number of the thymocyte population and induced the synthesis of low amounts of gamma-IFN. Preincubation of thymocytes with TPA increased the response to Con A; both the number of thymocytes expressing receptors and the synthesis of gamma-IFN were increased. Addition of IL 2 to these cultures further augmented the expression of IL 2 receptors and gamma-IFN synthesis and resulted in the optimal expression of IL 2 receptors and maximal gamma-IFN synthesis. The expression of IL 2 receptors could be detected within 24 hr and preceded the induction of proliferation; it was therefore probably not due to the clonal expansion of a population of receptor-bearing thymocytes. Conversely, inhibition of IL 2 synthesis with dexamethasone (Dex) by thymocytes activated with Con A, or inhibition of the function of IL 2 receptors by anti-Tac, resulted in a decrease in the number of IL 2 receptor-bearing thymocytes activated with Con A, or inhibition of the function of IL 2 receptors by anti-Tac, resulted in a decrease in the number of IL 2 receptor-bearing thymocytes and of gamma-IFN synthesis. Thymocytes activated with TPA and Con A were more resistant to the inhibitory effects of Dex on the expression of IL 2 receptors than thymocytes activated with Con A alone. Maximal inhibition of the expression of IL 2 receptors and of gamma-IFN synthesis was achieved as a result of the synergistic effect of anti-Tac with Dex. Therefore, when IL 2 was prevented from binding to the receptors, and IL 2 synthesis was inhibited, the number of thymocytes expressing IL 2 receptors was sharply reduced and gamma-IFN synthesis was markedly inhibited.(ABSTRACT TRUNCATED AT 400 WORDS)     

Thymic lymphocytes. III. Cooperative phenomenon in the proliferation of thymocytes under Con A stimulation

In the present paper, the response of thymocytes to Con A is analyzed in terms of a cooperative phenomenon between medullary thymocytes, cortical thymocytes, thymic accessory cells, and interleukin 2. Medullary thymocytes respond spontaneously to Con A and produce IL-2. The addition of exogenously produced IL-2 enhances their proliferation. Small numbers of cortical (PNA+) thymocytes do not respond to Con A, even in the presence of IL-2-containing supernatant. By increasing the number of PNA+ cells per well, sensitivity to Con A and IL-2 appears. This response may be linked either to the increase in a minor PNA+-responding population and/or to the enhanced contamination by medullary thymocytes and macrophages in non-responding PNA+ thymocyte population. In this hypothesis, either the contaminating cells respond by themselves and/or cooperate with PNA+ cells to induce their proliferation. Coculture of non-responding low numbers of PNA+ thymocytes with Con A- and IL-2-containing supernatant in the presence of PNA- cells containing thymic medullary thymocytes and macrophages always produces a higher response than that of each individual population. These results show that a cooperative phenomenon occurs in the cocultures of PNA+ and PNA- thymic cells. We can show using PNA+ and PNA- thymocytes with different Thy 1 alleles, that indeed both PNA+ and populations participate PNA-thymocytes with different Thy 1 alleles, that indeed both PNA+ and PNA- populations participate in the generation of proliferating cells. We can demonstrate, by lysis experiments with monoclonal antibodies and complement that at the end of coculture, most of the proliferating cells are Lyt 1+, and part are Lyt 2+ or L3T4+. We discuss the fact that the phenotype of the cells after activation does not allow us to deduce the phenotype of their precursors. Lysis of Ia+ cells prior to coculture, reduces the level of the proliferative response but does not modify the percentage of cooperation produced by the coculture. Cooperation with medullary mature thymocytes or the presence of active Ia- accessory cells possibly able to convert to Ia expression during coculture experiments may account for these results.     

Dexamethasone-induced phospholipase A2-inhibitory proteins (PLIP) influenced by the H-2 histocompatibility region

Recent work has indicated that the H-2 histocompatibility complex on chromosome 17 influences the degree of glucocorticoid-induced teratogenicity and anti-inflammatory response. Since both of these hormonal actions appear to be mediated by the induction of phospholipase A2-inhibitory proteins (PLIP), the influence of the H-2 complex on the induction of PLIP by glucocorticoids in thymocytes and embryonic palates has been investigated. Analysis of dexamethasone-induced PLIP by Sephadex G-100 revealed four peaks of mol wt 55,000, 40,000, 28,000 and 15,000 in mouse thymocytes and from one to three of these PLIPs in mouse embryonic palates. The 55,000 mol wt PLIP comprised 50-60% of the total activity. The total amount of dexamethasone-induced PLIP is significantly higher in B10.A (H-2a) thymocytes than that in thymocytes of their congenic resistant partners, B10 (H-2b). The induced level of PLIP in the embryonic palates treated with dexamethasone is also significantly higher in the H-2a congenic strains with either the A or B background (AWy or B10.A) than that in their resistant partners (A.BY or B10). Thus, both susceptibility to glucocorticoid-induced cleft palate and the production of PLIP by this hormone are influenced by the H-2 complex.     

Concanavalin A-induced translocation of part of the GTP-binding activity from the membrane to the cytosol in murine thymocytes

Concanavalin A (Con A) stimulation resulted in the rapid redistribution of part of the GTP-binding activity from the membrane to the cytosol in murine thymocytes. This change in GTP-binding activity was dependent on the Con A concentration. To investigate the relationship between this redistribution and phospholipase C (PLC) activity, the effect of GTP gamma S on the cytosol PLC activity was also examined, and it was found that GTP gamma S enhanced the phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis activity in the cytosol of Con A-stimulated thymocytes more than in that of unstimulated thymocytes. This enhancement by GTP gamma S was also dependent on the Con A concentration. The results suggest that in murine thymocytes, the GTP-binding protein (G-protein) involved in the regulation of PLC activity may be translocated from the membrane to the cytosol upon Con A stimulation. Besides, the dose dependence curve for the change in the GTP gamma S-binding activity was similar to that for inositol phosphates formation in Con A-stimulated thymocytes, suggesting that the translocation of the G-protein is closely related to PLC activation. Furthermore, the effects of cytosol fractions containing the 38-43 and 23-28 kDa GTP-binding subunits of G-proteins on the PIP2 hydrolysis activity of partially purified PLC were examined. The fraction containing the 23-28 kDa subunit evidently enhanced the PLC activity but that containing the 38-43 kDa subunit enhanced the activity to a much lower extent. Moreover, the 23-28 kDa subunit fraction of Con A-stimulated thymocytes was more effective as to enhancement of the PLC activity than that of unstimulated thymocytes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Spermine prevents cytochrome c release in glucocorticoid-induced apoptosis in mouse thymocytes

Apoptosis can be induced in primary cultures of mouse thymocytes using the glucocorticoid dexamethasone. Addition of the polyamine spermine simultaneously with dexamethasone reduces the induction of apoptosis compared to treatment with dexamethasone alone. We investigated the signal transduction pathway at the mitochondrial level in order to elucidate spermine's protective effect. Mitochondrial involvement is evident due to the loss of mitochondrial transmembrane potential, release of cytochrome c into the cytosol and activation of caspase-9 in dexamethasone-treated thymocytes. The addition of spermine inhibited the release of cytochrome c from the mitochondria into the cytosol, and also the activation of caspase-9. When the mitogen concanavalin A (Con A) was added to dexamethasone- plus spermine-treated thymocytes, the number of apoptotic cells in the pre-G(1)peak was reduced compared to thymocytes treated with only dexamethasone plus spermine. Comparing concanavalin A added to dexamethasone-treated or to dexamethasone plus spermine-treated thymocytes, showed a markedly reduced pre-G(1)peak in the latter. Thus, the spermine-induced inhibition of cytochrome c release confers a survival advantage on thymocytes.     

Selective activation of murine V beta 8.2 bearing T cells by Pseudomonas exotoxin A.

We have determined that Pseudomonas aeruginosa exotoxin A (PE) can selectively stimulate the proliferation of V beta bearing T lymphocytes. Murine thymocytes were fractionated by selective agglutination with peanut agglutinin (PNA) and the PNA- thymocytes, which represent mature thymocytes, were shown to be responsive to PE stimulation. In addition, mature peripheral T lymphocytes (nylon wool nonadherent splenocytes) were also observed to respond to PE stimulation. Both CD4+ and CD8+ splenic T lymphocyte populations proliferated in response to PE. Flow microfluorimetry analysis of PNA- thymocytes stimulated with PE indicated that V beta 8.2 bearing T cells were preferentially expanded. Thus, our data indicate that PE represents a microbial super antigen which stimulates murine thymocytes which bear the V beta 8.2 element of the T cell receptor.     

Cutting Edge: A possible role for CD4+ thymic macrophages as professional scavengers of apoptotic thymocytes

A vast majority of thymocytes are eliminated during T cell development by apoptosis. However, apoptotic thymocytes are not usually found in the thymus, indicating that apoptotic thymocytes must be eliminated rapidly by scavengers. Although macrophages and dendritic cells are believed to play such role, little is known about scavengers in the thymus. We found that CD4(+)/CD11b(+)/CD11c(-) cells were present in the thymus and that they expressed costimulatory molecules for T cell selection and possessed Ag-presenting activity. Moreover, these CD4(+)/CD11b(+) cells phagocytosed apoptotic thymocytes much more efficiently than thymic CD4(-)/CD11b(+) cells as well as activated peritoneal macrophages. CD4(+)/CD11b(+) cells became larger along with thymus development, while no such change was observed in CD4(-)/CD11b(+) cells. Finally, engulfed nuclei were frequently found in CD4(+)/CD11b(+) cells. These results strongly suggest that thymic CD4(+)/CD11b(+) cells are major scavengers of apoptotic thymocytes.     

A sensitive IL-1 thymocyte co-stimulator assay using a novel beta-D-galactoside specific lectin from the beetle, Allomyrina dichotoma

A sensitive thymocyte co-stimulator assay of IL-1 using a beta-D-galactoside specific lectin (allo A) obtained from the beetle (Allomyrina dichotoma) is reported here. Allo A stimulated [3H]thymidine uptake of mouse thymocytes in the presence of IL-1. The allo A assay was more sensitive than the PHA or PNA- thymocyte assay, especially at low doses of IL-1. Optimal conditions for the allo A assay were as follows: allo A, 2.5-5.0 micrograms/ml; whole thymocytes, 0.5-1.0 x 10(6) cells/well; incubation time, 72-96 hr. The assay is sensitive and convenient and can easily be performed in any laboratory.     

Functional diversity of polypeptides in primary culture supernatant of thymus epithelial cells

Polypeptide fractions A-C (M.W., 7 kd, 4.7 kd, and 3 kd) were obtained from the primary culture supernatant of thymus epithelial cells from Wistar rats by high-pressure liquid chromatography with a gel-filtration column. Changes in the mitogen responses of rat thymocytes and their subpopulations with addition of a fraction were studied. One subpopulation was rich in non-rosette-forming cells (non-RFCs), and the other was cortisone resistant thymocytes (CRTs). These subpopulations were incubated with a fraction for 24 hrs. before mitogen stimulation. Fractions A and C increased the response of the non-RFCs to concanavalin A (Con A) and that of total thymocytes and CRTs to phytohemagglutinin (PHA). Fraction B inhibited Con A and PHA response of total thymocytes and CRTs. Fraction B was cytotoxic toward total thymocytes and CRTs when viability was evaluated by [3H]uridine prelabelling. This cytotoxicity was suppressed by treatment with trypsin. Subfractions B3 and B4 obtained by reversed-phase column chromatography were cytotoxic toward CRTs. The effects of the fractions on thymocyte maturation were different, showing their functional diversity.     

Differences in the pattern of proliferative response with age in thymocytes undergoing spontaneous and induced proliferation

The proliferative capacity of thymocytes from C3H/HeJ mice decrease as the animals attain maturity. The proliferative response of thymocytes from 24- to 28-week-old mice to stimulation with concanavalin A (Con A) is only 20% of that observed at 4 weeks of age. The decreased proliferative capacity of thymocytes in response to Con A stimulation observed between 4 and 24 weeks of age closely correlates to the drop in thymic weight and cellularity observed during this period. In contrast, the spontaneous proliferative capacity of thymocytes, as well as proliferation of thymocytes in response to stimulation with phorbol myristate acetate (PMA) and ionomycin, drops only slightly during this period, as proliferation under these condition in thymocytes from 24- to 28-week-old mice is approximately 65-70% of that observed in 4-week-old animals. We have previously shown that cytoplasmic extracts from proliferating lymphoid cells contain a factor, termed the activator of DNA replication (ADR), which is capable of inducing DNA synthesis in isolated, quiescent nuclei. We show in this study that the decreased proliferative capacity of thymocytes during whole organism maturation and thymic involution is associated with decreased endogenous levels of ADR, while nuclear sensitivity of thymocyte to ADR was retained during these process. The diminution of ADR activity during thymic involution was quantitatively greater than the loss in proliferative capacity.