Interleukin 2-induced lymphocyte proliferation is independent of increases in cytosolic-free calcium concentrations

Activation of lymphocytes by mitogenic lectins initiates a sequence of events that culminates in DNA synthesis and cell proliferation. The mitogenic effects of lectins on T lymphocytes leads to the production of a group of lymphokines including the interleukins. The binding of interleukin 2 (IL 2) to its receptor results in activation of the cell leading to DNA synthesis. An increase in cytosolic-free Ca++ ([Ca++]i) is associated with activation of lymphocytes by mitogenic lectins and also appears to be a prerequisite for induction of DNA synthesis and cell proliferation. We have determined whether the proliferative response triggered by IL 2 binding to its receptor is associated with or requires an increase in [Ca++]i. Using human and murine IL 2-sensitive cell lines, we have demonstrated that the IL 2-induced proliferative response, in contrast to that induced by mitogens such as phytohemagglutinin or concanavalin A, is not accompanied by an increase in [Ca++]i as monitored by the fluorescent indicator quin-2. Furthermore, IL 2-dependent triggering of lymphoblasts occurs in the presence of extremely low extracellular calcium concentrations that prevent transmembrane calcium flux. Activation of IL 2 receptor-bearing T cells, therefore, does not appear to be associated with or to require an increase in [Ca++]i as part of the activation and signaling process. The critical step requiring calcium flux in cell signaling by mitogenic lectins must therefore occur elsewhere in the activation cascade.     

Cholera toxin-sensitive 3',5'-cyclic adenosine monophosphate and calcium signals of the human dopamine-D1 receptor: selective potentiation by protein kinase A.

The signal transduction pathways of the dopamine-D1 receptor were investigated in two cell types stably transfected with the human D1 receptor cDNA, rat pituitary GH4C1 cells (GH4-hD1), and mouse Ltk-fibroblast cells (L-hD1). In both GH4-hD1 and L-hD1 cell lines, stimulation of the dopamine-D1 receptor induced a marked increase in cAMP accumulation. In addition, dopamine potentiated activation of L-type voltage-dependent calcium channels in a cAMP-dependent manner in GH4-hD1 cells. However, in L-hD1 cells, dopamine increased cytosolic free calcium concentrations ([Ca++]i) by mobilization of intracellular calcium rather than by calcium influx. This effect was correlated with a dopamine-induced enhancement of phospholipase C activity in L-hD1 cells. Pretreatment (24 h) with cholera toxin (CTX) was used to maximally activate the GTP-binding protein (G protein) Gs, causing a maximal elevation of cAMP levels and uncoupling the D1 receptor from Gs. The described actions of dopamine in both cell lines were abolished by pretreatment with CTX, indicating that CTX substrates (e.g. Gs) may mediate these actions. The blockade by CTX was not due to CTX-induced elevation of cAMP, since pretreatment with forskolin or 8-bromo-cAMP to activate cAMP-dependent protein kinase did not inhibit dopamine actions nor alter basal [Ca++]i. Pretreatment (1-3 h) of L-hD1 cells with forskolin (10 microM) or 8-bromo-cAMP (5 mM) altered neither the basal activity of phospholipase C nor basal [Ca++]i in L-hD1 cells but greatly enhanced the dopamine-induced increase of phosphatidyl inositol turnover and [Ca++]i. From these results we conclude that: 1) the dopamine-D1 receptor induces multiple and cell-specific signals, including elevation of cAMP levels in both GH and L cells, cAMP-dependent activation and potentiation of opening of L-type voltage-dependent calcium channel in GH cells, and a novel phosphatidyl inositol-linked mobilization of cellular calcium in L cells; 2) coupling of the D1 receptor to these responses involves CTX-sensitive proteins, possibly Gs; and 3) acute preactivation of cAMP-dependent protein kinase can markedly enhance, rather than attenuate, certain pathways of dopamine-D1 transmembrane signaling.     

Extracellular calcium ion depletion in frog cardiac ventricular muscle.

The extracellular free [Ca++] in frog ventricular muscle strips was monitored using single-barrel calcium ion-selective microelectrodes. During trains of repetitive stimulation, a heart rate-dependent, sustained fall (depletion) of the extracellular free [Ca++] occurs, which is most likely a consequence of net Ca++ influx into ventricular cells. The magnitude of the [Ca++]0 depletion increases for higher Ringer's solution [Ca++], and is reversibly blocked by manganese ion. Prolonged repetitive field stimulation (20-30 min) activates additional cellular Ca++ efflux, which can balance the additional Ca++ influx caused by stimulation, resulting in abolition of extratrabecular [Ca++]0 depletion in 20-30 min, and hence zero net transmembrane Ca++ flux at steady state. In the poststimulation period of quiescence, cellular Ca++ efflux persists and causes an elevation (accumulation) of the extracellular free [Ca++]. From these [Ca++]0 depletions, quantitative estimates for the net transmembrane Ca++ flux were derived using an analytical solution to the diffusion equation. In the highest Ringer's solution [Ca++] used (1 mM) the calculated net increase of the total intracellular calcium per beat was 6.5 +/- 1.4 mumol/l of intracellular space. This corresponds to an average net transmembrane Ca++ influx of 0.81 +/- 0.17 pmol/cm2/s during the 800-ms action potential. In lower bath [Ca++] the net transmembrane [Ca++] flux was proportionately reduced.     

Characterization of the requirements for human T cell mitogenesis by using suboptimal concentrations of phytohemagglutinin

To characterize the requirements for T cell proliferation, we have studied the response of purified populations of human T cells to varying concentrations of the mitogen phytohemagglutinin (PHA). Concentrations of PHA which induce optimal proliferative responses induce increases in cytosolic free calcium ([Ca2+]i), expression of interleukin 2 (IL 2) receptors, and production of IL 2. As the concentration of PHA is decreased, each of these processes decreases in parallel. At suboptimal concentrations of PHA, the addition of exogenous IL 2 reconstitutes both the proliferative response and the expression of the IL 2 receptor, as measured by immunofluorescence with antibodies directed against the TAC/IL 2 receptor molecule, but without reconstituting the increase in [Ca2+]i. Therefore, the concentration dependence of responses to PHA appears to be secondary to an absence of IL 2 production due to a failure to induce an increase in [Ca2+]i. The addition of the calcium ionophores A23187 and ionomycin or of accessory cells to low concentrations of PHA induces increases in [Ca2+]i and subsequent proliferative responses, suggesting that the two events are linked. The proliferative response can be inhibited by antibodies directed towards IL 2 or the IL 2 receptor, indicating that the proliferative response was at least partially dependent on the production and action of IL 2. This suggests that, although increases in [Ca2+]i are an integral event in the induction of proliferation by PHA, the increase in [Ca2+]i is required for the production but not the action of IL 2. In addition, low concentrations of PHA deliver an additional signal to cells, independent of an increase in [Ca2+]i, which induces IL 2 receptor expression and allows a proliferative response in the presence of exogenous IL 2.     

Antigen-specific and -nonspecific mitogenic signals in the activation of human T cell clones

We have directly compared the signals required for: induction of the [Ca+2]i response, expression of Tac antigen, and proliferation in antigen-specific human T cell clones. We have previously shown that antigen-specific activation of cloned T cells under conditions leading to proliferation is accompanied by a rapid increase in [Ca+2]i. Cloned T cells showed increased [Ca+2]i, enhanced Tac expression, and proliferated in response to specific antigen in the presence of viable, genetically appropriate antigen-presenting cells. Paraformaldehyde fixation of antigen-presenting cells after "pulsing" with antigen prevented proliferation, but did not affect MHC-restricted [Ca+2]i or Tac responses. Treatment of cloned T cells with monoclonal anti-T3 antibody also increased [Ca+2]i and Tac expression but did not induce proliferation. Proliferation was restored by viable autologous or allogenic APC or exogenous IL 2, but not by IL 1. In contrast to resting T cells, T cell clones were insensitive to the mitogenic effects of lectins or of ionophores and phorbol esters. These results suggest that activation of antigen-specific T cells requires the sequential action of at least two signals. The first is MHC restricted and is mediated by interaction of antigen + MHC class II products with the T cell receptor (T3-Ti) complex. This leads to Tac expression and increased [Ca+2]i, but is not sufficient for proliferation. This signal can be bypassed by anti-T3 monoclonal antibodies. Proliferation requires a second, nonantigen-specific, non-MHC-restricted antigen-presenting cell signal, which cannot be replaced by IL 1 in our system. This signal can be bypassed, however, by the addition of exogenous IL 2 to cells that have received the first signal and express Tac, suggesting that it is required for IL 2 synthesis and secretion. T cell clones therefore provide a useful model for studying antigen-dependent and -independent events in cell activation.     

Role of membrane potential in the response of human T lymphocytes to phytohemagglutinin

We have analyzed the role of membrane potential on T cell activation and cell proliferation. Depolarization of T lymphocytes, by increasing the extracellular concentration of K+ during a 1-hr exposure to PHA, results in a marked inhibition of cell proliferation. In parallel, depolarization of T cells prevented the normal increase in [Ca2+]i seen after PHA binding. In depolarized cells, PHA failed to induce IL 2 secretion, but, in contrast, IL 2 receptor expression was triggered normally and the cells were subsequently responsive to exogenous IL 2. Increasing [Ca2+]i in depolarized cells with the ionophore ionomycin, or bypassing the requirement for an increase in [Ca2+]i with TPA, restored the PHA-induced proliferative response in depolarized cells. These data confirm that a membrane potential-sensitive step, namely, Ca2+ influx and the resulting change in [Ca2+]i, is triggered by PHA. The inhibitory effects of depolarization are mediated through the impairment of IL 2 secretion, but not IL 2 receptor expression. T cell proliferation can therefore be regulated by altering membrane potential, which in turn modulates the extent of the change in [Ca2+]i. This study suggests a role for transmembrane potential in the regulation of the T cell proliferative response.     

Requirement for extracellular calcium or magnesium in mitogen-induced activation of human peripheral blood lymphocytes

The importance of calcium in lymphocyte activation is well recognized, but the levels of extracellular ionized free calcium (Ca++) necessary for lymphocyte proliferation via various pathways have not been investigated in detail. We studied the ability of a lectin mitogen (PHA) and a calcium ionophore (ionomycin) to induce interleukin 2 receptors, interleukin 2 (IL2) production, and proliferation over various concentrations of extracellular Ca++. Reducing the Ca++ levels from the normal 200 microM to 10 microM in PHA-stimulated cultures partially inhibited IL2 receptor expression, IL2 production, and subsequent proliferation. At 1 microM Ca++, both IL2 activity and proliferation were eliminated, but partial IL2 receptor expression was still observed. Ionomycin did not induce any of these events in cultures where the extracellular Ca++ concentration was below 100 microM. Restoring calcium in the medium resulted in normal levels of IL2 receptor expression, IL2 activity, and proliferation when PBL were stimulated with either mitogen. Exogenous magnesium partially restored these events in PHA-stimulated cultures, but had no effect when ionomycin was used as the mitogen. These data indicate that stimulation by ionomycin is much more dependent upon the levels of extracellular Ca++ than is PHA. Extracellular calcium also appears to be necessary subsequent to IL2 receptor acquisition, since the latter was seen without IL2 activity or proliferation at very low extracellular Ca++, and IL2 failed to restore the proliferative response under these conditions. The data also suggest that PHA, but not ionomycin, can activate lymphocytes via a magnesium-dependent pathway, or that PHA has a lower specificity for divalent cation cofactors.     

Molecular mechanisms involved in T cell activation. III. The role of extracellular calcium in antigen-induced lymphokine production and interleukin 2-induced proliferation of cloned cytotoxic T lymphocytes

The role that extracellular calcium plays in activating resting cloned cytotoxic T lymphocytes (CTL) to proliferate and to produce lymphokines was examined. In these cells, stimulation with interleukin 2 (IL-2) induced a proliferative response without a concomitant production of macrophage-activating factor (MAF), whereas stimulation with antigen or lectin (in the absence of IL-2) induced MAF production but not proliferation. In the case of IL-2-induced proliferation, extracellular calcium was required to initiate proliferation as well as to prevent cellular arrest later in the G2 + M phase of the cell cycle. In MAF production extracellular calcium was required both to activate the phosphatidylinositol signal-transducing mechanism and to mobilize intracellular calcium in antigen- or lectin-stimulated cytotoxic T lymphocytes. Further, extracellular calcium was required for only 8 of the 18 hr of stimulation time which was needed to achieve maximal MAF production, indicating that both calcium-dependent and -independent events exist in the signal pathway. Additional experiments with calcium ionophores and activators of protein kinase C indicated that although both intracellular calcium mobilization and de novo protein phosphorylation are involved in MAF production, an optimal increase in the level of intracellular calcium by itself is insufficient to induce the production of this lymphokine.     

Intracellular free calcium in rat anterior pituitary cells monitored by fura-2

Rat anterior pituitary cells, loaded with the calcium indicator dye fura-2 after primary culture, were challenged with prolactin and growth hormone secretagogues and inhibitory hormones. To initially validate the technique, the calcium channel activator maitotoxin effectively increased intracellular free calcium [( Ca++]i). Various concentrations of the secretagogues thyrotropin releasing hormone or angiotensin II induced peak increases in [Ca++]i within 15 sec, followed by a lower and prolonged plateau phase. The inhibitory hormones dopamine and somatostatin maximally reduced [Ca++]i by 15-20 sec, followed by a spontaneous return to baseline over 5-10 min. The receptor antagonists saralacin and spiperone blocked the angiotensin II and dopamine effects, respectively. Thus, fura-2 appears to be an adequate probe for resolving second-to-second changes in [Ca++]i induced by hormone receptor activation in anterior pituitary cells.     

An intracellular calcium increase and protein kinase C activation fail to initiate T cell proliferation in the absence of a costimulatory signal

Resting T lymphocytes proliferate in response to a combination of a calcium ionophore and a phorbol ester. This observation suggests that an increase in intracellular calcium free ion concentration [Ca2+]i and activation of protein kinase C (PKC) are sufficient signaling events for the initiation of T cell proliferation. In contrast, an accessory cell-generated costimulatory signal, acting independently of the rise in [Ca2+]i and PKC activation, is required for Ag-induced proliferation of type I T cell clones. We now report that this costimulatory signal is unexpectedly also being delivered via a cell-cell interaction during the response to ionomycin and phorbol ester. In the absence of this signal (at limiting cell numbers), T cells fail to divide. We also demonstrate that proliferation in response to immobilized anti-CD3 mAb requires the cell-cell interaction. These results suggest a model of T cell stimulation in which activation of a costimulatory signaling pathway is important in the regulation of the IL-2 gene, and only in the presence of this (third) signal can an increase in [Ca2+]i and PKC activity induce T cell proliferation. Such a model predicts that IL-2-dependent expansion of T cell clones in vivo in response to Ag receptor occupancy requires the delivery of an independent accessory cell-derived co-stimulatory signal.