Antigen-dependent increase in cytosolic free calcium in specific human T-lymphocyte clones

Calcium has been implicated as an intracellular messenger in the cellular response to various external stimuli. Exposure of lymphocytes to various mitogens and lectins results in rapid transmembrane calcium fluxes and increased cytoplasmic calcium concentrations ([Ca2+]i). It is not clear, however, whether the mechanisms by which these non-physiological stimuli activate cells are related to those involved in antigen-specific activation. We have now used antigen-specific T-cell clones to study changes in [Ca2+]i associated with specific activation and show here that these cells respond specifically in the presence of antigen and antigen-presenting cells (APC) with increased [Ca2+]i and that this increased [Ca2+]i shows the same genetic restrictions as are seen in the proliferation assay. The kinetics of the [Ca2+]i response to antigen indicate that antigen undergoes a time-dependent processing step as a prerequisite for recognition by T cells, as has been shown for T-cell proliferative responses, but that the [Ca2+]i response to processed antigen is extremely rapid. The close correlation between changes in [Ca2+]i and cell activation resulting in proliferation suggests that Ca2+ may act as an intracellular messenger in antigen-specific responses.     

Thy-1 functions as a signal transduction molecule in T lymphocytes and transfected B lymphocytes

Thy-1, a glycoprotein of relative molecular mass 25,000 (25K), is a major constituent of the cell surface of mouse thymocytes, peripheral T cells and neurones. In man, Thy-1 is present on neurones and on a small percentage of thymocytes, but is absent from peripheral T cells. The amino-acid and complementary DNA sequences of Thy-1 indicate that it has a structure similar to an isolated V (variable region) domain of immunoglobulin. Although the function of Thy-1 is unknown, the ability of different anti-Thy-1 monoclonal antibodies to activate murine T cells or induce functional changes in neuronal cells in vitro suggests that Thy-1 is involved in transmembrane signalling. We now show that crosslinking of murine Thy-1 triggers a rapid rise in the cytoplasmic free calcium concentration ([Ca2+]i), not only in murine T cells and Thy-1.2-transfected human T cells, but also in murine B-lymphoma cells transfected with the murine thy-1.2 gene. These results indicate that the generation and transduction of the signal leading to the rise in [Ca2+]i is independent of the T-cell receptor and other T-cell-specific molecules. The preservation of the [Ca2+]i-modulating function of Thy-1 in various lymphoid cells of two species further suggests that the necessary signal either originates in the Thy-1 molecule itself or is generated in concert with a highly conserved molecules(s) associated with Thy-1.     

Thy-1-mediated T-cell activation requires co-expression of CD3/Ti complex

In addition to monoclonal antibodies against the CD3 (T3)-T-cell antigen receptor (CD3/Ti) complex, several other monoclonals directed towards distinct cell surface structures on human (CD2 (T11) and Tp44) and murine (Thy-1, TAP, and Ly-6) T lymphocytes are capable of activating T cells. It has been proposed that such structures may function as alternative pathways of stimulation. To examine directly whether any relationship exists between Thy-1-dependent activation phenomena and T-cell activation mediated through the CD3/Ti complex, we have transfected several CD3/Ti- variants of the human T-cell line Jurkat with the murine Thy-1.2 gene. Our data indicate that in CD3/Ti-, Thy-1.2+ transfectants, monoclonal antibodies against Thy-1.2 can induce a rise in cytoplasmic free calcium ([Ca2+]i), but fail to stimulate interleukin-2 (IL-2) production. The only defect in these variant cell lines responsible for the inability to produce IL-2 in response to Thy-1 stimulation was in the expression of the CD3/Ti complex, because replacement of defective Ti alpha- or beta-chain genes reconstributed both surface expression of CD3/Ti and responsiveness to Thy-1 in the IL-2 production assay.     

Activation of cytolytic T lymphocyte and natural killer cell function through the T11 sheep erythrocyte binding protein

The T11 sheep erythrocyte binding glycoprotein [relative molecular mass (Mr)50,000(50K)] is expressed throughout human T-lymphocyte ontogeny and appears to play an important physiological role in T-cell activation. Thus, the treatment of T cells with certain monoclonal anti-T11 antibodies results in antigen-independent polyclonal T-cell activation as assessed by proliferation and lymphokine secretion. In addition, the majority of thymocytes that have not yet acquired the T3-Ti antigen/major histocompatibility complex (MHC) receptor can be activated to express interleukin-2 (IL-2) receptors through this T11 structure. We show here that the triggering of cytolytic T (Tc) cells via T11 causes an antigen-independent activation of the cytolytic mechanism as evidenced by the induction of nonspecific cytolytic activity. Furthermore, T11+T3-Ti- natural killer (NK) cell clones can also be induced to lyse NK-cell-resistant targets by treatment with anti-T11 monoclonal antibodies directed at defined T11 epitopes. These results indicate that T11 triggering can activate cytotoxic lymphocytes to express their functional programmes in the absence of specific antigen recognition via the T3-Ti complex and provide further evidence for the notion that certain NK cells and T lymphocytes are related.     

Heterotrimeric G protein participated in modulation of cytoplasmic calcium concentration in pollen cells

Cytoplasmic free calcium concentration([Ca2+]c) in pollen cells of Lilium daviddi is measured with confocal laser scanning microscopy to investigate the effect of heterotrimeric G protein (G protein) on [Ca2+]c and the possible signal transduction pathway of G protein triggering cellular calcium signal. After application, cholera toxin (CTX), an agonist of G protein, triggers a transient increase of [Ca2+]c in pollen cells, and evokes a spatial-temporal characteristic calcium dynamics; while pertussis toxin (PTX), a G protein antagonist, leads to the decrease of [Ca2+]c. Both L-type Ca2+ channel blocker verapamil and inhibitor of IP3 receptor heparin inhibit CTX-induced [Ca2+]c increase. The results show that G protein may play a role in the modulation of [Ca2+]c through enhancing the extracellular Ca2+ influx and releasing of Ca2+ from intracellular stores.     

Physiological [Ca2+]i level and pump-leak turnover in intact red cells measured using an incorporated Ca chelator

The physiological actions of Ca2+ as a trigger and second messenger depend on the maintenance of large inward resting Ca2+ gradients across the cell plasma membrane. An ATP-fuelled Ca-pump, originally discovered and still best characterized in human red cells, is now believed to mediate resting Ca2+ extrusion in most animal cells. However, even in red cells, the truly physiological pump-leak turnover rate and cytoplasmic free Ca2+ level are unknown. Previous estimates were only very imprecise upper limits because normal intact red cells have a minute total pool of exchangeable Ca of less than 1 mumol 1 cells; Ca fluxes could not be measured without artificially increasing that pool with ionophores or disrupting the membrane to incorporate Ca buffers. Both procedures leave the membrane considerably leakier than in intact cells. Here, we have increased the exchangeable Ca pool by non-disruptively loading a Ca-chelator into intact cells, using intracellular hydrolysis of a membrane-permeant ester. The trapped chelator made the free cytoplasmic calcium concentration, [Ca2+]i, an easily defined function of directly measurable total cell Ca. We were then able to establish the physiological steady-state [Ca2+]i and pump-leak turnover rate of fresh cells suspended in their own plasma. If [Ca2+]i was lowered below the normal resting level, the Ca pump rate decreased according to the square of [Ca2+]i, and the inward Ca leak increased. The increase in leak did not develop if the cells were depleted of ATP and ADP.     

Dissociation of phosphoinositide hydrolysis and Ca2+ fluxes from the biological responses of a T-cell hybridoma

T lymphocytes can be activated in a variety of ways, including occupancy of the T cell antigen receptor (TCR) complex or cross-linking of certain cell-surface molecules with antibody. Two of the earliest events seen after stimulation are the hydrolysis of phosphatidylinositol bisphosphate to inositol trisphosphate (Ins P3) and 1,2-diacylglycerol (DAG), and an increase in the concentration of intracellular Ca2+ ([Ca2+]i). Later, the cell secretes lymphokines and expresses lymphokine receptors. It has been postulated that the products of the hydrolysis of phosphatidylinositols (Ptd Ins) and fluctuations in [Ca2+]i are critical 'second messengers', transmitting the signals for the initiation of the later events. We have examined the relationship between these second messengers and the secretion of IL-2 in a murine T cell variant whose missing TCR complex had been reconstituted by gene transfer. Surprisingly, although the IL-2 responses of the transfectant could not be distinguished from the original line expressing the same TCR, Ptd Ins hydrolysis and the increase in [Ca2+]i were substantially reduced or absent in the reconstituted cell. It is therefore possible to dissociate these early biochemical changes from a late biological response, raising questions about the putative causal relationship of these events.     

Single-channel and whole-cell recordings of mitogen-regulated inward currents in human cloned helper T lymphocytes

Cytoplasmic free Ca2+ [( Ca2+]i) appears to be an important signal for DNA synthesis in early stages of lymphocyte activation. In spite of many experimental studies which employ fluorescent Ca2+ indicator dye to demonstrate an early increase of [Ca2+]i in T-lymphocytes after stimulation with lectins, specific antigens, and monoclonal antibodies to T-lymphocyte receptors, the mechanism responsible for the rise of [Ca2+]i is unknown. We have used the extracellular patch clamp technique to investigate this mechanism. Unitary inward currents, mediated by Ca2+ or Ba2+, were recorded in the membrane of T-lymphocytes. The inward current channel was characterized by a conductance of 7 pS and extrapolated reversal potential (Erev) 110 mV positive to resting potential (Vr). While gating kinetic parameters were not affected by membrane potential changes, the probability of channel opening markedly increased upon activation of the T-lymphocyte by the mitogenic lectin, phytohaemagglutinin (PHA). PHA also evoked a cadmium-sensitive, inward Ba2+ current on whole-cell clamp. We suggest that this mitogen-regulated channel introduces Ca2+ into the cytoplasm upon activation and represents a new class of voltage-independent Ca2+ channels.     

Ion channels in human neutrophils activated by a rise in free cytosolic calcium concentration

A rapid, transient rise in the free cytosolic Ca2+ concentration ([Ca2+]i) is one of the earliest events in neutrophil activation and is assumed to be involved in many of the subsequent cellular reactions. Both Ca2+ release from intracellular stores and Ca2+ influx from the extracellular space contribute to the rise in [Ca2+]i. In an attempt to assess the relative importance of these pools and the sequences leading to the rise in [Ca2+]i, we have studied the time course of changes in [Ca2+]i after stimulation with N-formyl-methionyl-leucyl-phenylalanine (fMLP) or platelet-activating factor (PAF) using the Ca2+ indicators quin-2 and fura-2. We observed a time lag of 1-3 s between stimulation and rise in [Ca2+]i. This lag depends on the agonist concentration but is independent of extracellular Ca2+. Thus Ca2+ release from intracellular stores is rate limiting for the rise in [Ca2+]i. After this, cation channels in the plasma membrane (measured with the patch clamp method) are opened. These non-selective channels, which also pass Ca2+, are activated by the initial rise in [Ca2+]i, but by neither fMLP nor inositol 1,4,5-trisphosphate (IP3) directly.     

The T lymphocyte glycoprotein CD2 binds the cell surface ligand LFA-3

CD2 (known also as T11 (ref. 1), LFA-2 (ref. 2) and the erythrocyte rosette receptor (ref. 3] is a functionally important T lymphocyte surface glycoprotein of relative molecular mass 50,000 to 58,000 (Mr 50-58 K) which appears early in thymocyte ontogeny and is present on all mature T cells. Monoclonal antibodies to CD2 inhibit cytotoxic T-lymphocyte (CTL)-mediated killing by binding to the T lymphocyte and blocking adhesion to the target cell. Such antibodies also inhibit T helper cell responses including antigen-stimulated proliferation, interleukin-2 (IL-2) secretion, and IL-2 receptor expression. Certain combinations of monoclonal antibodies to CD2 epitopes trigger proliferation of peripheral blood T lymphocytes, cytotoxic effector function and expression of IL-2 receptors by thymocytes, resulting in thymocyte proliferation in the presence of exogenous IL-2 (ref. 11). These findings suggest that CD2 can function in signalling as well as being an adhesion molecule. To understand the role of CD2 in T-cell adhesion and activation, it is essential to define its natural ligand. Our previous observation that purified CD2 inhibits rosetting of T lymphocytes with sheep erythrocytes and can be absorbed by sheep erythrocytes suggested it also might bind with detectable affinity to human cells. We now report that CD2 binds to a cell-surface antigen known as lymphocyte function-associated antigen-3 (LFA-3) with high affinity, and can mediate adhesion of lymphoid cells via interaction with LFA-3.     

Oscillations of intracellular Ca2+ in mammalian cardiac muscle

Contraction of cardiac muscle depends on a transient rise of intracellular calcium concentration ([Ca2+]i) which is initiated by the action potential. It has, however, also been suggested that [Ca2+]i can fluctuate in the absence of changes in membrane potential. The evidence for this is indirect and comes from observations of (1) fluctuations of contractile force in intact cells, (2) spontaneous cellular movements, and (3) spontaneous contractions in cells which have been skinned to remove the surface membrane. The fluctuations in force are particularly prominent when the cell is Ca2+-loaded, and have been attributed to a Ca2+-induced Ca2+ release from the sarcoplasmic reticulum. In these conditions of Ca2+-loading the normal cardiac contraction is followed by an aftercontraction which has been attributed to the synchronization of the fluctuations. The rise of [Ca2+]i which is thought to underlie the aftercontraction also produces a transient inward current. This current, which probably results from a Ca2+-activated nonspecific cation conductance, has been implicated in the genesis of various cardiac arrhythmias. However, despite the potential importance of such fluctuations of [Ca2+]i their existence has, so far, only been inferred from tension measurements. Here we present direct measurements of such oscillations of [Ca2+]i.     

Inhibition of mitosis by an antibody to the mitotic calcium transport system

Calcium ions are important in the regulation of mitotic apparatus assembly and in the control of chromosome movement. Changes in intracellular free calcium concentration, [Ca2+]i are achieved by an intracellular calcium-transport system which is highly conserved in different cell types. A membrane-bound protein of relative molecular mass (Mr) 46,000 (46K) is part of this transport system and has been implicated in the regulation of the [Ca2+]i changes associated with the course of mitosis. A monoclonal antibody against this 46K protein inhibits Ca2+-uptake into isolated Ca2+-sequestering membranes and specifically labels membranes associated with the mitotic apparatus of sea urchin embryos. Here we investigate the relationship between the intracellular calcium transport system and mitosis by injection of this monoclonal antibody into living mitotic sea urchin embryos. We find that after injection the intracellular free calcium increases up to 10(-6) M, the mitotic apparatus is rapidly destroyed and the cell is irreversibly blocked in its development.     

Cell-to-cell spread of calcium signals mediated by ATP receptors in mast cells.

Rat basophilic leukaemia cells, like mast cells from which they are derived, have surface Fc epsilon receptors that trigger secretion of inflammatory mediators when crosslinked. Both GTP-binding proteins and a rise in cytosolic calcium concentration ([Ca2+]i) are implicated in the secretory mechanism. Here we use a video-imaging technique to report that transient rises in [Ca2+]i initiated in an individual cell can spread from cell to cell in a wave-like pattern by means of a secreted intermediate, in the absence of gap-junctional communication. We find that the leukaemia cells, peritoneal mast cells and mucosal mast cells have cell-surface P2-type purinergic receptors that can trigger similar [Ca2+]i transients. We provide evidence that ATP is rapidly released, and that it can amplify [Ca2+]i signals and initial secretory responses during antigen-stimulation of rat basophilic leukaemia cells.     

Kinetics, stoichiometry and role of the Na-Ca exchange mechanism in isolated cardiac myocytes

Compelling evidence has existed for more than a decade for a sodium/calcium (Na-Ca) exchange mechanism in the surface membrane of mammalian heart muscle cells which exchanges about three sodium ions for each calcium ion. Although it is known that cardiac muscle contraction is regulated by a transient increase in intracellular calcium ([Ca2+]i) triggered by the action potential, the contribution of the Na-Ca exchanger to the [Ca2+]i transient and to calcium extrusion during rest is unclear. To clarify these questions, changes in [Ca2+]i were measured with indo-1 in single cardiac myocytes which were voltage clamped and dialysed with a physiological level of sodium. We find that Ca entry through the Na-Ca exchanger is too slow to affect markedly the rate of rise of the normal [Ca2+]i transient. On repolarization, Ca extrusion by the exchanger causes [Ca2+]i to decline with a time constant of 0.5 s at -80 mV. The rate of decline can be slowed e-fold with a 77-mV depolarization. Calcium extrusion by the exchanger can account for about 15% of the rate of decline of the [Ca2+]i transient (the remainder being calcium resequestration by the sarcoplasmic reticulum (SR]. The ability of the cell to extrude calcium was greatly reduced on inhibiting the exchanger by removing external sodium, which itself led to an increase in resting [Ca2+]i. This finding is in contrast to the suggestion that calcium extrusion at rest is mediated mainly by a sarcolemmal Ca-ATPase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Intracellular free calcium rise triggers nuclear envelope breakdown in the sea urchin embryo

Cytosolic free calcium has recently been implicated in the regulation of mitosis in plant and animal cells. We have previously found correlations between increases in the levels of intracellular free calcium [Ca2+]i and visible transitions of structure at nuclear envelope breakdown (NEBD) and the onset of anaphase during mitosis in sea urchin embryos and tissue culture cells. To go beyond correlations it is necessary to manipulate [Ca2+]i, and in sea urchin embryos this requires the injection of calcium-chelator buffer solutions as the changes in free calcium in the cell cycle are dependent on intracellular stores. We report here that blocking the increase in [Ca2+]i which just precedes NEBD prevents this from taking place and halts mitosis. Subsequent injections which momentarily increase [Ca2+]i, or a natural recovery of the higher calcium levels, result in NEBD and the successful continuation of mitosis. Similarly, artificially increasing calcium by early injections results in early NEBD. We conclude that the increase in [Ca2+]i preceding NEBD is an essential regulatory step required for entry into mitosis.     

Role for microsomal Ca storage in mammalian neurones?

Alterations in the intracellular concentration of calcium ions [( Ca2+]i) are increasingly being found to be associated with regulatory functions in cells of all kinds. In muscle, an elevation of [Ca2+]i is the final link in excitation-contraction coupling while at nerve endings and in secretory cells, similar rises in [Ca2+]i are thought to mediate exocytosis. The discovery of calcium-activated ion channels indicated a role for intracellular calcium in the regulation of membrane excitability. Calcium transients associated with either intracellular release or the inward movement of Ca2+ across the membrane have been recorded in molluscan neurons and more recently in neurones of bullfrog sympathetic ganglia. Here, we report the first recordings of calcium transients in single mammalian neurones. In these experiments we have found that the methylxanthine, caffeine, causes the release of calcium from a labile intracellular store which can be refilled by Ca2+ entering the cell during action potentials.     

Expression and function of CD4 in a murine T-cell hybridoma

The CD4 (T4) antigen was originally described as a phenotypic marker specific for helper T cells, and has recently been shown to be the receptor for the human immunodeficiency virus (HIV). Functional studies using monoclonal antibodies directed at CD4 and major histocompatibility complex (MHC) class II molecules led to the suggestion that CD4 binds to the MHC class II molecules expressed on stimulator cells, enhancing T-cell responsiveness by increasing the avidity of T cell-stimulator cell interaction and/or by transmitting a positive intracellular signal. But recent evidence that antibodies to CD4 inhibit T-cell responsiveness in the absence of any putative ligand for CD4 has been interpreted as suggesting that antibody-mediated inhibition may involve the transmission of a negative signal via the CD4 molecule instead. We have infected a murine T-cell hybridoma that produces interleukin 2 (IL-2) in response to human class II HLA-DR antigens with a retroviral vector containing CD4 cDNA. The resulting CD4-expressing hybridoma cell lines produce 6- to 20-fold more IL-2 in response to HLA-DR antigens than control cell lines. Furthermore, when antigen levels are suboptimal, the response of the cell lines is entirely CD4-dependent. The data presented here clearly demonstrate that CD4 can enhance T-cell responsiveness and may be crucial in the response to suboptimal levels of antigen.     

Ca2+ signals induced from calcium stores in pancreatic islet β cells

In single rat pancreatic β cells,using fura-2 microfluorometry to measure [Ca2+]i response upon different stimuli,the ways of calcium regulation have been studied.When the extracellular calcium concentration was 2.5 mmol/L,either 60 mmol/L KCl,20 mmol/L D-glucose or 0.1 mmol/L tolbutamide induced increase in [Ca2+]i.Such increase in [Ca2+]i was absent when the same stimuli were applied under zero extracellular calcium.These results indicate that the increase of [Ca2+]i is induced by the activation of voltage-dependent calcium channels in β cells.The manifold forms of [Ca2+]i change induced by glucose imply that the effects of glucose are complex.5 mmol/L caffeine or 5 mmol/L MCh increase the [Ca2+]i ,which is independent of the external calcium,suggesting that [Ca2+]i can be regulated by Ca2+ release from not only the IP3-sensitive but also the ryanodine sensitive calcium stores in β cells.The latency of Ca responses for IP3 pathway (5 s) is faster than that for ryanodine pathway (30 s).It is concluded that there are multiple calcium stores in rat pancreatic β cells.     

Mitogens trigger a calcium-independent signal for proliferation in phorbol-ester-treated lymphocytes

The activation of T lymphocytes by mitogens requires at least two signals; the first, delivered to T cells by a mitogen in conjunction with accessory cells (monocytes/macrophages), leads to the generation of the second signal, interleukin-2 (IL-2). The first signal also induces the expression of IL-2 receptors on the surface of a subpopulation of T cells; binding of IL-2 to its receptor then initiates a cascade of events culminating in DNA synthesis by these cells. Certain compounds act synergistically with mitogens in promoting T-cell proliferation by substituting for the activities of interacting cells or their products. For example, the phorbol ester 12-O-tetradecanoyl phorbol 13-acetate (TPA) has been shown to restore the ability of macrophage-depleted T-cell populations to respond to mitogenic lectins. Transmembrane fluxes of calcium, leading to increased free cytosolic calcium concentrations ([Ca2+]), have been demonstrated following mitogen binding to lymphocytes and have been implicated in the initiation of cell proliferation. We show here that the effect of TPA on lymphocyte proliferation occurs in the absence of extracellular Ca2+ or detectable changes in [Ca2+]i, but only in the presence of mitogens. This suggests that in cells which have been incubated with the phorbol ester, mitogens can induce proliferation by a calcium-independent signal.     

Protein kinase C activation of physiological processes in human neutrophils at vanishingly small cytosolic Ca2+ levels

It has long been assumed that a rise in cytosolic free Ca2+, [Ca2+]i, is a necessary and sufficient event for the stimulation of a variety of cellular processes. The development of a technique which allows monitoring of [Ca2+]i in small intact cells has led to a critical revision of this simple postulate. We have recently shown that in neutrophils, Ca2+-ionophore-induced elevations of [Ca2+]i, quantitatively similar to those caused by chemotatic peptides, are ineffective in stimulating cell responses, which suggests that an additional signal is required for receptor-mediated activation. Here we show that subthreshold concentrations of phorbol myristate acetate (PMA) and of a Ca2+ ionophore can quantitatively mimic the effect of a physiological agonist. However, PMA at higher concentrations can trigger NADPH-oxidase activity, exocytosis and protein phosphorylation, even when [Ca2+]i is lowered 10-20 times below the normal resting level. These results strongly suggest that activation of protein kinase C is sufficient, by itself, to induce NADPH-oxidase activation and exocytosis of secondary granules in neutrophils.