Inhibition of DNA fragmentation in thymocytes and isolated thymocyte nuclei by agents that stimulate protein kinase C

Glucocorticoid hormones and Ca2+ ionophores stimulate a suicide process in immature thymocytes, known as apoptosis or programmed cell death, that involves extensive DNA fragmentation. We have recently shown that a sustained increase in cytosolic Ca2+ concentration stimulates DNA fragmentation and cell killing in glucocorticoid- or ionophore-treated thymocytes. However, a sustained increase in the cytosolic Ca2+ level also mediates lymphocyte proliferation, suggesting that apoptosis is blocked in proliferating thymocytes. In this study we report that phorbol esters, which selectively stimulate protein kinase C (PKC), blocked DNA fragmentation and cell death in thymocytes exposed to Ca2+ ionophore or glucocorticoid hormone. The T cell mitogen, concanavalin A, which stimulates thymocytes by a mechanism that involves PKC activation, caused concentration-dependent increases in the cytosolic Ca2+ level that did not result in DNA fragmentation, but incubation with concanavalin A and the PKC inhibitor H-7 (1-(5-isoquinolinylsulfonyl)-2-methylpiperazine) resulted in both DNA fragmentation and cell death. Phorbol ester directly inhibited Ca2+-dependent DNA fragmentation in isolated thymocyte nuclei. Our results strongly suggest that PKC activation blocks thymocyte apoptosis by preventing Ca2+-stimulated endonuclease activation.     

Nonthermal 60 Hz sinusoidal magnetic-field exposure enhances 45Ca2+ uptake in rat thymocytes: dependence on mitogen activation

The effect of a 60 Hz sinusoidal magnetic field of nonthermal intensity on Ca2+ metabolism in rat thymic lymphocytes (thymocytes) was assessed in resting cells and in cells activated with the mitogen Concanavalin A (Con A). A 60 min exposure at 37 degrees C to an induced electric field of 1.0 mV/cm produced an average 2.7-fold increase in Con A-dependent 45Ca2(+)-uptake compared to non-exposed, isothermal control cells. In contrast, 45Ca2+ uptake remained unaltered during exposure of resting thymocytes. It was also found that thymocytes with a diminished ability to mobilize Ca2+ in response to Con A were most sensitive to the 60 Hz magnetic field. Although the precise mechanism of field interaction is at present unknown, modulation of Ca2+ metabolism during cell activation may represent a common pathway for field coupling to cellular systems.     

Charybdotoxin-sensitive, Ca(2+)-dependent membrane potential changes are not involved in human T or B cell activation and proliferation.

The involvement of ion channels in B and T lymphocyte activation is supported by many reports of changes in ion fluxes and membrane potential after mitogen binding. Human T and B lymphocytes demonstrate an early and transient hyperpolarization after ligand binding. Inasmuch as the change in membrane potential is dependent on elevation of free cytosolic calcium, the hyperpolarization is presumably through opening of Ca(2+)-stimulated K+ channels. We have used charybdotoxin, a known inhibitor of Ca(2+)-dependent K+ channels, to study the role of these channels in lymphocyte activation and mitogenesis. We demonstrate that charybdotoxin inhibits the ligand-induced transient membrane hyperpolarization in B and T cells in a dose-dependent fashion, without affecting changes in cytosolic Ca2+. However, blockade of the Ca(2+)-activated K+ channel is not associated with changes in cell-cycle gene activation, IL-2 production, IL-2R expression or B and T cell mitogenesis. These results imply that membrane potential changes secondary to the ligand-dependent opening of Ca(2+)-activated K+ channels are not involved in B and T lymphocyte activation and mitogenesis.     

17 beta-estradiol increases Ca(2+) influx and down regulates interleukin-2 receptor in mouse thymocytes

The influx of Ca(2+) across the T lymphocyte membrane is an essential triggering signal for activation and proliferation by an antigen. The aim of this study was to determine if Ca(2+) influx through estradiol receptor (ER) operated channels of Ca(2+) entry induced activation of lymphoid cells. Mouse thymocytes were incubated with 17 beta-estradiol (E) and in the presence or absence of the mitogen, phytohemagglutinin (PHA). Despite evidence of an enhanced binding of E to ER on thymocyte membranes, and an E dose-related influx of Ca(2+), there was a consistent down regulation of IL-2 receptor expression (P < 0.001). Incubation of thymocytes with PHA enhanced IL-2 receptor expression although the down regulatory effect of E was still evident. The results suggest that the Ca(2+) channel activated by E may have a down regulatory effect on the IL-2 receptor in thymus cells leading to the dampening of cell activation process.     

Beta-endorphin modulation of mitogen-stimulated calcium uptake by rat thymocytes

Lymphocytes stimulated by mitogens or antigens exhibit an enhanced calcium uptake early in the proliferation or activation response. Modulation of this calcium uptake results in alterations of proliferation and immunocompetence. beta-endorphin and other opioids affect several parameters of lymphocyte competence. Limited data are available concerning the mechanism(s) of these effects. This study examines whether a possible opioid mechanism is the modification of the early calcium influx into stimulated lymphocytes. The time course of both concanavalin A (Con A) and phytohemagglutinin (PHA)-stimulated 45Ca2+ uptake into thymocytes was characterized to determine the optimal time for testing the effects of opioids. beta-Endorphin 1-31 significantly enhanced Con A-stimulated 45Ca2+ uptake into rat thymocytes. This peptide had no significant effect on PHA-stimulated 45Ca2+ uptake or on basal thymocyte 45Ca2+ flux. The beta h-endorphin stimulatory effect was titratable in the range of 0.1 nM to 10 microM. Naloxone did not reverse the enhancement. Met-enkephalinamide and other opioid agonists did not duplicate the stimulatory effect. Thus, the beta h-endorphin 1-31 enhancement of Con A-stimulated 45Ca2+ uptake by rat thymocytes does not operate via classical opioid receptor mechanisms. beta h-endorphin 1-31 appears to be acting on a subset of T cells that are responsive to Con A but not to PHA.     

Inhibitors of arachidonic acid metabolism eliminate the increase in cytosolic free calcium induced by the mitogen concanavalin A in rat thymocytes

Using inhibitors of arachidonic acid (AA) metabolism, the possible involvement of AA products in the generation of [Ca2+]i and the pHi rise induced by the mitogen concanavalin A (Con A) in rat thymocytes has been studied. The lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA, 10 microM) and the phospholipase A2 inhibitor bromophenacyl bromide (10 microM) eliminated the [Ca2+]i signal induced by Con A; the cyclooxygenase blocker indomethacin also inhibited it. However, neither NDGA nor indomethacin suppressed the pHi rise stimulated by Con A. Exogenous AA induced an increase in [Ca2+]i but not in the pHi. These results indicate that AA metabolites, probably of the lipoxygenase pathway, take part in the generation of the [Ca2+]i response to the mitogen. In contrast, they appear not to be involved in the pHi rise evoked by Con A.     

Changes in Ca2+ level in cytoplasm of rat thymocytes after treatment with mitogens

Mitogen-induced changes in free Ca2+ concentration in the cytoplasm [Cai2+] of rat thymocytes were studied with the use of quin-2, a Ca2+-sensitive fluorescent indicator. Concanavalin A (Con A) and phytohemagglutinin were shown to increase [Cai2+] from 150 +/- 10 nM for the resting cells up to the value of 380 +/- 10 nM. This increase in [Cai2+] depended on the mitogen concentration. It was observed both in the presence of 1 mM external Ca2+ and in the Ca2+ free medium. The Con A-induced increase of [Cai2+] was not abolished by Na+ removal from the medium or by verapamil, an inhibitor of potential-dependent Ca2+ channels. Hence, the increase in Cai2+ was not due to an activation of potential operated Ca2+-channels. Agents which raise intracellular cAMP blocked Con A-induced increase of [Cai2+].     

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.     

Accelerated calcium ion uptake in murine thymocytes induced by concanavalin A.

The mechanism of enhancement of Ca2+ uptake by the T cell mitogen concanavalin A (Con A) was studied in murine thymocytes. Native Con A enhanced the rate of Ca2+ uptake as much as 9-fold, an increase being observed within five minutes after Con A addition. The effect of Con A was reversed completely by alpha-methyl mannopyranoside (alpha-MM). Increased Ca2+ uptake was observed with increasing concentrations of Con A, between 2 and 400 microgram/ml, indicating that the stimulation of Ca2+ uptake is not restricted to mitogenic lectin concentrations (0.5-2 microgram/ml). Succinyl Con A exhibits only a slight effect in the same concentration ranges as native Con A. Ca2+ uptake, both in the absence and presence of Con A, is strongly dependent on energy metabolism and is carrier mediated. The augmentation of Ca2+ uptake by native Con A is due to an enhanced Vmax. Uptake of the anion, CrO42-, by thymocytes, found to be a non-saturable process, was also enhanced by Con A. The effect of Con A on CrO42- permeability appears to be independent of its effect on Ca2+ uptake.     

The relationship between mitogen-induced changes in the cytoplasmic pH and free Ca2+ concentration in rat thymocytes

The relationship between pHi and [Ca]i signals generated in rat thymocytes by the mitogen Con A has been investigated. It is shown that the mitogen-induced [Ca]i rise is dependent on Na+/H+ exchange or some other Na(+)-sensitive process. This conclusion is based on the following findings: (i) [Ca]i response to Con A weakens upon decreasing the concentration of extracellular Na+, or inhibiting Na+/H+ exchange; (ii) agents that alkalinize the cytoplasm (the phorbol ester TPA, the Na+/H+ ionophore monensin and NH4Cl) cause an increase in [Ca]i (Klip, A., Rothstein, A. and Mack, E. (1984) Biochem. Biophys. Res. Commun. 124, 14-22; Grinstein, S. and Goetz, J.D. (1985) Biochim. Biophys. Acta 819, 267-270); (iii) The effects of Con A, TPA and monensin on [Ca]i are not additive. The last observation suggests that all these agents activate the same Na+/H+ (Na+ and/or H+)-dependent system of Ca2+ transport. It is found that the pH i and [Ca]i responses in rat thymocytes are sensitive to changes in the intracellular levels of cyclic nucleotides, ATP and in temperature. These regulatory effects on the ionic signals are different for Con A, TPA and monensin. In particular, both the stimulation of Na+/H+ antiport and the [Ca]i rise brought about by Con A or TPA are inhibited upon elevating the cellular cAMP. In contrast, the monensin-induced [Ca]i signal is almost independent of cAMP but is highly sensitive to changes in cGMP and temperature. Reducing the ATP level eliminates both the pHi and [Ca]i responses to Con A but not to monensin. These different characteristics of [Ca]i signals elicited by the mitogen and the Na+/H+ ionophore indicate that these agents use different mechanisms to activate the Na+/H(+)-dependent Ca2+ transporting system. A [Ca]i response to monensin has been obtained in some other cell types, namely, in lymphoblastoid Raji cells, Ehrlich ascites tumor cells and also in platelets.     

Opioid agonist modulation of cytoplasmic free Ca2+ level in concanavalin A-stimulated mouse lymphocytes.

In this study the influence of mu-, delta-, and kappa-selective opioid agonists (DAMGO, DSLET, and dynorphin A (1-13)) on cytoplasmic free Ca2+ ([Ca2+]i) level in normal and concanavalin-A (Con A)-activated mouse lymphocytes was investigated. [Ca2+]i was measured using the fluorescent dye FURA-2AM. The opioid peptides at 10-12-10-7 M induced some increase in [Ca2+]i in non-activated lymphocytes. However, DAMGO and DSLET (10-13-10-7 M) considerably inhibited a Con A-induced increase in [Ca2+]i. The inhibiting effect of both peptides was higher after 20-min preincubation compare to 2-h preincubation. The effect of the kappa-agonist dynorphin A (1-13) was significantly different depending on the duration of cell pretreatment and the concentration of the peptide used. After preincubation for 20 min at low concentrations (10-12-10-11 M) it slightly stimulated, while at higher (10-10-10-7 M) concentrations it inhibited lymphocyte response to Con A. After preincubation for 2 h, pronounced stimulation of mitogen-induced Ca2+ flux was observed at peptide concentration 10-9 M. The effects of opioids were antagonized by naloxone. These data indicate that functionally active opioid receptors expressed on lymphocytes could be involved in early stages of mitogen activation.     

The role of calcium ions in the regulation of rat thymocyte pyruvate oxidation by mitogens.

1. Calcium concentrations in the nanomolar range cause a specific stimulation of the oxidation of pyruvate by isolated mitochondria from rat thymus that is sufficient to account precisely for the stimulation of pyruvate oxidation observed when rat thymocytes are incubated with the mitogens concanavalin A or ionophore A23187. 2. Higher concentrations of Ca2+ (more than 50 nM) inhibit the oxidation of NAD+-linked substrates by rat thymus mitochondria without affecting the oxidation of succinate or ascorbate+ NNN'N'-tetramethyl-p-phenylendiamine. 3. The addition of Ni2+ or Co2+ (2mM) to rat thymocytes prevents the response to concanavalin A at the level of pyruvate oxidation without affecting the stimulation of glycolysis induced by this mitogen. In contrast, the complete metabolic response to the ionophore A23187 is abolished by these ions. Ni2+ and Co2+ interfere with the ability of the ionophore to transport Ca2+ across the plasma membrane. 4. Concanavalin A, but not ionophore A23187, increases the respiratory inhibition induced by Ni2+ and Co2+. 5. These results support the view that mitogens stimulate lymphocyte pyruvate oxidation through an increase in cellular Ca2+ uptake.     

Ca2+-dependent K+ transport in lymphocytes

The treatment of rat thymocytes with A23187 + Ca2+, ascorbate-phenazine methosulphate or propranolol induced quinine-sensitive fluxes of K+ (Rb+) suggesting the presence in the cell membrane of Ca2+-dependent K+ channels. Concanavalin A induced K+ channel activation only at very high doses (13 micrograms/ml). Neither quinine nor the increase of the K+ concentration in the medium to 30 mM prevented the stimulation of amino acid transport induced by concanavalin A, suggesting that the Ca2+-dependent K+ channel is not involved in the early phenomena of lymphocyte activation.     

Redistribution of intracellular Ca2+ in mitogen-stimulated human peripheral blood lymphocytes

The fluorescent probe chlortetracycline (CTC) was used to investigate redistribution of intracellular Ca2+ in concanavalin A (Con A)-stimulated human peripheral blood lymphocytes. The addition of the mitogen to CTC-equilibrated lymphocytes induced (within 10 to 15 minutes) a Con A-concentration dependent decrease in CTC fluorescence indicating the release of membrane-bound Ca2+. The effect was independent of the level of extracellular Ca2+ and could be observed in the presence of EGTA; it was suppressed by the metabolic inhibitors FCCP, antimycin and sodium cyanide. Analysis of the excitation spectra of CTC fluorescence indicated that the observed effect is caused by redistribution of intracellular Ca2+ rather than Mg2+. Thus the lectin interaction with the lymphocyte plasma membrane results in Ca2+ release into the cytosol from the intracellular stores.     

Properties of different Ca2+ pools in permeabilized rat thymocytes

The regulation of free Ca2+ concentration by intracellular pools and their participation in the mitogen-induced changes of the cytosolic free Ca2+ concentration, [Ca2+]i, was studied in digitonin-permeabilized and intact rat thymocytes using a Ca2+-selective electrode, chlortetracycline fluorescence and the Ca2+ indicator quin-2. It is shown that in permeabilized thymocytes Ca2+ can be accumulated by two intracellular compartments, mitochondrial and non-mitochondrial. Ca2+ uptake by the non-mitochondrial compartment, presumably the endoplasmic reticulum, is observed only in the presence of MgATP, is increased by oxalate and inhibited by vanadate. The mitochondria do not accumulate calcium at a free Ca2+ concentration below 1 microM. The non-mitochondrial compartment has a greater affinity for calcium and is capable of sequestering Ca2+ at a free Ca2+ concentration less than 1 microM. At free Ca2+ concentration close to the cytoplasmic (0.1 microM) the main calcium pool in permeabilized thymocytes is localized in the non-mitochondrial compartment. Ca2+ accumulated in the non-mitochondrial pool can be released by inositol 1,4,5-triphosphate (IP3) which has been inferred to mediate Ca2+ mobilization in a number of cell types. Under experimental conditions in which ATP-dependent Ca2+ influx is blocked, the addition of IP3 results in a large Ca2+ release from the non-mitochondrial pool; thus IP3 acts by activation of a specific efflux pathway rather than by inhibiting Ca2+ influx. SH reagents do not prevent IP3-induced Ca2+ mobilization. Addition of the mitochondrial uncouplers, FCCP or ClCCP, to intact thymocytes results in no increase in [Ca2+]i measured with quin-2 tetraoxymethyl ester whereas the Ca2+ ionophore A23187 induces a Ca2+ release from the non-mitochondrial store(s). Thus, the data obtained on intact cells agree with those obtained in permeabilized thymocytes. The mitogen concanavalin A increases [Ca2+]i in intact thymocytes suspended in both Ca2+-containing an Ca2+-free medium. This indicates a mitogen-induced mobilization of an intracellular Ca2+ pool, probably via the IP3 pathway.     

Defective thymic T cell activation by concanavalin A and anti-CD3 in autoimmune nonobese diabetic mice. Evidence for thymic T cell anergy that correlates with the onset of insulitis.

In nonobese diabetic (NOD) mice, T cells play a major role in mediating autoimmunity against pancreatic islet beta-cells. We and others previously reported that age-related alterations in the thymic and peripheral T cell repertoire and function occur in prediabetic NOD mice. To study the mechanism responsible for these T cell alterations, we examined whether a defect exists in the thymus of NOD mice at the level of TCR-mediated signaling after activation by Con A and anti-CD3. We found that thymocytes from NOD mice respond weakly to Con A- and anti-CD3-induced proliferation, compared with thymocytes from control BALB/c, BALB.B, (BALB.B x BALB.K)F1, C57BL/6, and nonobese non-diabetic mice. This defect correlates with the onset of insulitis, because it can be detected at 7 to 8 weeks of age, whereas younger mice displayed a normal T cell responsiveness. Thymic T cells from (NOD x BALB/c)F1 mice, which are insulitis- and diabetes-free, exhibit an intermediate stage of unresponsiveness. This T cell defect is not due to a difference in the level of CD3 and IL-2R expression by NOD and BALB/c thymocytes, and both NOD CD4+ CD8- and CD4- CD8+ mature thymic T cells respond poorly to Con A. BALB/c but not NOD thymic T cells respond to Con A in the presence of either BALB/c or NOD thymic APC, suggesting that the thymic T cell defect in NOD mice is intrinsic to NOD thymic T cells and is not due to an inability of NOD APC to provide a costimulatory signal. The defect can be partially reversed by the addition of rIL-2 to NOD thymocytes. To determine whether a defect in signal transduction mediates this NOD thymic T cell unresponsiveness, we tested whether these cells elevate their intracellular free Ca2+ ion concentration in response to Con A. An equivalent Con A-induced increase in Ca2+ ion concentration in both NOD and BALB/c thymocytes was observed, suggesting a normal coupling between the CD3 complex and phospholipase C in NOD thymocytes. In contrast to their low proliferative response to Con A or anti-CD3, NOD thymocytes respond normally (i.e., as do BALB/c thymocytes) to the combinations of PMA plus the Ca2+ ionophore ionomycin and PMA plus Con A but weakly to Con A plus ionomycin. Our data suggest that the age-related NOD thymocyte unresponsiveness to Con A and anti-CD3 results from a defect in the signaling pathway of T cell activation that occurs upstream of protein kinase C activation.     

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.     

5C6-F4, a novel 100,000-dalton rat lymphocyte activation antigen defined by monoclonal antibody

Con A-activated rat thymocytes were used to immunize mice, and immune spleen cells were fused with NS/1 myeloma cells. One clone, designated 5C6-F4, reacted strongly with Con A-activated rat thymocytes and some LPS-activated rat spleen cells but not with normal thymocytes, spleen cells, or bone marrow cells of rat origin. The 5C6-F4 did not react with Con A-activated thymocytes of mouse origin. Immunoprecipitation of 5C6-F4 antigen from surface-iodinated Con A-activated rat thymocytes or LPS-activated rat spleen cells revealed its m.w. to be approximately 100,000. The kinetic studies of the expression of 5C6-F4 antigen revealed that 5C6-F4 antigen was detectable at 6 hr after Con A stimulation of rat spleen cells, whereas IL 2 receptor (IL 2R) was detectable at 12 hr. The appearance of 5C6-F4 antigen and IL 2R precede the onset of DNA synthesis of Con A-activated spleen cells. Thus, 5C6-F4 antigen is classified as early activation antigen. The 5C6-F4 inhibits the lymphocyte proliferation induced by mitogen and the IL 2-driven rat T cell proliferation. Sequential immunoprecipitation study as well as binding inhibition study indicated that the 5C6-F4 antigen is distinct from IL 2R molecule. The 5C6-F4 antigen appears to be a novel rat lymphocyte activation antigen that exhibits immunoregulatory function and also may serve as a useful marker of T cell activation.     

Calcium-dependent killing of immature thymocytes by stimulation via the CD3/T cell receptor complex

Development of tolerance to self Ag occurs during a negative cell selection process in the thymus. This selection process is thought to involve interactions between Ag-specific thymocyte receptors and self Ag presented by the MHC proteins on accessory cells, resulting in deletion of potentially harmful self-reactive precursors. However, the mechanisms underlying this clonal deletion have not been identified. In confirmation of previous findings (C. A. Smith, G. T. Williams, R. Kingston, E. J. Jenkins, and J. J. T. Owen, 1989. Antibodies to CD3/T-cell receptor complex induce death by apoptosis in immature T cells in thymic cultures. Nature 337:181), we have found that an anti-CD3 antibody stimulated DNA fragmentation, characteristic of a suicide mechanism known as apoptosis or programmed cell death (PCD), in suspensions of human thymocytes. Endonuclease activation and cell killing were dependent on an early, sustained increase in cytosolic Ca2+ concentration, most of which was of extracellular origin. Although the magnitude and duration of the Ca2+ increase were similar to those observed in response to Con A, the mitogen did not stimulate DNA fragmentation or cell death. Phorbol ester prevented Ca2+-dependent DNA fragmentation and cell killing in response to anti-CD3 or other agents that stimulated PCD, suggesting that activation of protein kinase C abrogated cell suicide. Disappearance of CD4+CD8+ immature thymocytes was generally observed in response to all agents that stimulated PCD, whereas mature PBL were insensitive to stimulation of PCD. Our results suggest that antibody-mediated stimulation of immature thymocytes via the TCR complex results in Ca2+-dependent, endonuclease-mediated cell killing, depending on the activation status of protein kinase C.     

Calcium regulates inositol 1,3,4,5-tetrakisphosphate production in lysed thymocytes and in intact cells stimulated with concanavalin A.

Lysed mouse thymocytes release [3H]inositol 1,4,5 trisphosphate from [3H]inositol-labelled phosphatidyl inositol 4,5-bisphosphate in response to GTP gamma S, and rapidly phosphorylate [3H]inositol 1,4,5-trisphosphate to [3H]inositol 1,3,4,5-tetrakisphosphate. The rate of phosphorylation is increased approximately 7-fold when the free [Ca2+] in the lysate is increased from 0.1 to 1 microM, the range in which the cytosolic free [Ca2+] increases in intact thymocytes in response to the mitogen concanavalin A. Stimulation of the intact cells with concanavalin A also results in a rapid and sustained increase in the amount of inositol 1,3,4,5-tetrakisphosphate, and a much smaller transient increase in 1,4,5-trisphosphate. Lowering [Ca2+] in the medium from 0.4 mM to 0.1 microM before addition of concanavalin A reduces accumulation of inositol 1,3,4,5-tetrakisphosphate by at least 3-fold whereas the increase in inositol 1,4,5-trisphosphate is sustained rather than transient. The data imply that in normal medium the activity of the inositol 1,4,5-trisphosphate kinase increases substantially in response to the rise in cytosolic free [Ca2+] generated by concanavalin A, accounting for both the transient accumulation of inositol 1,4,5-trisphosphate and the sustained high levels of inositol 1,3,4,5-tetrakisphosphate. Inositol 1,3,4,5-tetrakisphosphate is a strong candidate for the second messenger for Ca2+ entry across the plasma membrane. This would imply that the inositol polyphosphates regulate both Ca2+ entry and intracellular Ca2+ release, with feedback control of the inositol polyphosphate levels by Ca2+.