Con A-induced Increase Research Articles

Taxol protects the microtubules of concanavalin A-activated lymphocytes from disassembly by methylmercury, but DNA synthesis is still inhibited

We have shown previously that there is a good correlation between the degree of microtubule disassembly by methylmercury (MeHg) and the extent of inhibition of DNA replication in Concanavalin A (Con A)-stimulated mouse splenic lymphocytes. The purpose of this study was to determine if these two events are causally related and to examine the effects of MeHg-induced microtubule disassembly on earlier events of the stimulation process. We show that early steps constituting the activation pathway, such as the Con A-induced increase in Ca 2+ influx and the expression of interleukin 2 receptor, are not inhibited by concentrations of MeHg that disassemble microtubules. RNA synthesis is not affected by short-term (3 h) treatment with MeHg, but longer treatment (24 h) inhibits RNA synthesis. In contrast, DNA synthesis is effectively inhibited by a 3-h treatment with MeHg. In lymphocytes treated with taxol, microtubules are not disassembled by MeHg; however, the inhibition of RNA and DNA synthesis persists. We conclude that the inhibition of nucleic acid synthesis by MeHg is not causally related to MeHg-induced microtubule disassembly.

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.

Tumor necrosis factor as an activator of human granulocytes. Potentiation of the metabolisms triggered by the Ca2+-mobilizing agonists.

TNF stimulated superoxide (O2-) release directly in human granulocytes in a dose-dependent manner (1 to 1000 U/ml), although its potency was weak. TNF-induced O2- release was inhibited by cAMP agonists or ionomycin, and was not accompanied with an increase in cytoplasmic free Ca2+ [( Ca2+]i) and membrane potential changes (depolarization). These findings indicate that neither Ca2+ mobilization nor membrane depolarization is required for TNF-receptor-mediated cell activation. The pretreatment of human granulocytes with TNF enhanced O2- release and membrane depolarization in parallel stimulated by the receptor-mediated Ca2+-mobilizing agonists (FMLP, Con A, and wheat germ agglutinin) or the Ca2+ ionophore ionomycin, but not by PMA, a direct activator of protein kinase C. The optimal effect was obtained by pretreatment of cells with 100 U/ml TNF for 5 to 10 min at 37 degrees C, although the magnitude of enhancement varied according to the agonists used as subsequent stimuli. TNF did not affect an increase in [Ca2+]i stimulated by the Ca2+-mobilizing agonists, except Con A. Con A-induced increase in [Ca2+]i was enhanced by TNF in a dose-dependent manner. These diverse effects of TNF could be partly explained by the exclusive potentiation by TNF of the metabolic events triggered by an increase in [Ca2+]i.

Stimulation of respiration by mitogens in rat thymocytes is independent of mitochondrial calcium.

The role of calcium in the control of respiration by the mitogen concanavalin A (ConA) was investigated in rat thymocytes. ConA induced an increase in both mitochondrial respiration and the mitochondrial calcium pool. The stimulation of respiration was shown to be independent of the increase in mitochondrial calcium: the calcium pool declined after 3 min, whereas the respiration increase was persistent, and was not affected by depletion of the calcium pool or by buffering intracellular Ca2+ transients with quin2. The mitogen phytohaemagglutinin stimulated respiration to the same extent as ConA, but did not increase the mitochondrial calcium pool. In addition, respiration was unaffected by changes in the mitochondrial calcium pool induced by increasing or decreasing extracellular calcium. These results indicate that control of respiration is not located in the Ca2+-sensitive mitochondrial dehydrogenases. The ConA-induced increase in respiration could be blocked by oligomycin, suggesting control by cytoplasmic ATP turnover, and was not associated with detectable changes in NAD(P)H fluorescence, indicating a balance between increased electron transfer and increased supply of reduced substrates.

Regulation of the number of Na+,K+-pump sites after mitogenic activation of lymphocytes.

The early activation of Na+,K+-ATPase-mediated ion fluxes after concanavalin A (ConA) stimulation of pig lymphocytes is caused by an increase in intracellular Na+ concentration. A second mechanism of regulation of Na+,K+-ATPase activity becomes apparent between 3 and 5 h after mitogenic stimulation, but prior to onset of increase in cell volume; this consists of an increase (about 75%) in the number of ouabain-binding sites (from 35 X 10(3) +/- 12 X 10(3)/cell in resting to 60 X 10(3) +/- 27 X 10(3)/cell in activated lymphocytes). The increase in ouabain binding was attributed to an increase in the number of active Na+,K+-ATPase molecules, based on the following evidence: there was an increase in the Vmax of ouabain binding, without variation in the Km; the increase in ouabain binding was accompanied by a proportional increase in K+ influx, when the assay was performed in the presence of the Na+ ionophore monesin, which was used to eliminate the difference in intracellular Na+ concentration between resting and activated cells; there was proportionality between ouabain-inhibitable ATPase activity in permeabilized cells and the number of ouabain-binding sites in resting and activated lymphocytes. The ConA-induced increase in ouabain-binding sites was influenced neither by amiloride nor by incubation in low Na+ medium, under conditions which prevented both increase in intracellular Na+ concentration and K+ influx. Increase in intracellular Na+ concentration was ineffective in altering the number of active pump molecules in resting cells. During incubation with ConA, the presence of ouabain did not affect the increase in ouabain-binding sites; thus, regulation of the number of pump sites is independent of the regulation of their activity. The ConA-induced increase in number of ouabain-binding sites did not require protein synthesis; indeed, cycloheximide, anisomycin, and puromycin, under conditions in which they inhibited protein synthesis by by 95%, induced the increase to approximately the same extent as did ConA. This suggests the presence in resting lymphocytes of a rapidly turning over protein that either prevents the ATPase subunits from assembling or from integrating into the membrane.

Sodium ion influx in proliferating lymphocytes: An early component of the mitogenic signal

Stimulation of pig peripheral blood lymphocytes with concanavalin A (Con A) provoked a rapid increase (two- to threefold) in the rate of ouabain-inhibitable K + uptake observable within 3–10 min of stimulation with mitogen. At least two phases can be distinguished in the activation of the Na + K + pump: the early phase (till 3 h) is characterized by an unaltered number of ouabain binding sites and the later phase (noted at 5 h) by an increased number of such sites. Both K + efflux and influx increased to the same extent, thereby maintaining [K +] i at the same level as in resting cells (120 m m). Within 3 min of addition of mitogen, the rates of total and amiloride-inhibitable Na + uptake went up two- and fourfold, respectively, thus resulting in rapid increase in [Na +] i from 20 to about 50 m m. Activation of the Na + K + pump was not observed when the cells were stimulated with Con A in low Na + medium (9 m m), nor did the usual rise in [Na +] i occur. When monensin (30 μ m), a Na + H + ionophore, was added to resting cells, an increase in both [Na +] i and active K + uptake occurred in normal medium but not when cells were suspended in low Na + isotonic buffer. Amiloride (500 μ m), on the other hand, prevented both the Con A-induced increase in [Na +] i and the activation of the Na + K + pump. Despite complete inhibition of the Na +,K +-ATPase in the presence of ouabain (1 mM), Con A activated the amiloride-inhibitable Na + uptake in the usual way. In mouse splenocytes stimulated with Con A, there was also a parallel rise in both [Na +] i and active K + uptake but this took considerably longer to occur than was the case in pig peripheral blood lymphocytes. Increase in both ionic fluxes, the former passive and the latter active, is essential to the entry and maintenance of the cells in proliferative cycle.

The effect of colchicine on cholesterol biosynthesis in concanavalin A-stimulated lymphocytes

Attempting to test the hypothesis that colchicine blocks lymphocyte activation at the commitment point, we determined whether or not colchicine inhibited the ConA-induced increase in the synthesis and content of cholesterol in lymphocyte cultures. Colchicine decreased the incorporation of ( 14C) acetate into cholesterol by about 50%. However, the decrease affected unstimulated and stimulated lymphocytes equally. The presence of colchicine for as long as 48 hours did not prevent stimulated lymphocytes from accumulating cholesterol. The results suggest that an intact microtubular system is not required for the initiation of blastogenesis and the activation of cholesterol synthesis.

Effect of Concanavalin A on Intracellular K + and Na + Concentration and K + Transport of Human Lymphocytes

Incubation of human lymphocytes with ConA causes an increase in [Na +]; and a decrease in [K +];. This effect is not due to the experimental washing procedure, but is due to the ConAinduced increase in permeability which is not fully compensated by the increase in active transport. The ConA-induced increase in 42K + uptake consists of an increase in leak flux which is independent of [Na +] o, and of an increase in pump flux which is dependent on [Na +] 0. The increase in leak flux may be caused by increased membrane fluidity. The increase in pump flux may be produced by the increased [Na +]. and by a stimulation of Na +, K + ATPase.

The cellular nature of concanavalin A-stimulated antibody-dependent cell-mediated cytotoxicity.

Enhanced antibody-dependent cytotoxic cell (ADCC) activity, generated in vitro by concanavalin A (Con A) stimulation of human peripheral blood lymphocytes, was analyzed to identify the nature of the killer (K) cell. Stimulated and unstimulated 2-day cultured cells were separated into sheep erythrocyte rosetting (E+) and non-rosetting (E-) subpopulations, and ADCC activity was determined by using the slopes of linear dose-response plots as a measure of K cell activity. Quantitative comparisons were made by examining activity on a per cell basis ("specific" activity) and total K cell activity recovered per subpopulation. This analysis revealed that although the E- cells were enriched for specific ADCC activity, the E+ subpopulation contained the major proportion of the total recovered activity and nearly all of the Con A-induced increase in K cell activity.

Early Nuclear Events in the Induction of Lymphocyte Proliferation by Mitogens: EFFECTS OF CONCANAVALIN A ON THE PHOSPHORYLATION AND DISTRIBUTION OF NON-HISTONE CHROMATIN PROTEINS

Abstract The mitogen concanavalin A (Con A) initiates a series of alterations of chromatin proteins early after addition to cultures of purified equine lymphocytes. Within 15 min of addition, Con A induces cellular non-histone proteins to bind to the chromatin and stimulates the phosphorylation of specific nuclear acidic proteins. The rate of phosphorylation of phenol-soluble nuclear acidic proteins increases as much as 4-fold within 2 hours of Con A stimulation. Phosphorylation is maximal 8 hours after Con A addition. The amount of phenol-soluble non-histone protein associated with the DNA increases as much as 4-fold within 6 hours of Con A stimulation. Con A also stimulates the synthesis of nuclear acidic proteins. Cycloheximide inhibits nuclear acidic protein synthesis by more than 90% but does not appreciably diminish the Con A-induced increase in the amount of these proteins entering the nucleus. Prelabeling of lymphocytes with [14C]leucine followed by treatment with Con A in unlabeled medium results in increased labeling of nuclear acidic proteins, indicating that presynthesized proteins are induced to bind to the chromatin in the presence of Con A. Experiments performed with lymphocytes fractionated by nonaqueous procedures indicate that proteins associating with the chromatin in response to Con A are largely of cytoplasmic origin. These results suggest that both a flux of specific proteins from the cytoplasm to the nucleus and an increase in phosphorylation of nuclear acidic proteins precede the extensive gene activation promoted by mitogens.