Sign-reversal during persistent activation in mu-opioid signal transduction

A concept of signal transduction in biological systems specifies that any instantaneous input is appreciated by its departure from the moving average of past activity. The concept provides an adequate account of the occurrence of both the one-directional (e.g. analgesic) effects induced by opioid receptor activation, and of the contra-directional (e.g. hyperalgesic) effects that can be observed when activation is discontinued. Following this transduction concept, the numerical simulations reported here revealed, remarkably, that under some parametric conditions, the input's effect may reverse even as input is maintained at a constant magnitude. In in vitro conditions that are proximal to the signal transduction that occurs when an opioid agonist binds to the G-protein coupled opioid receptor, the effects of opioid receptor activation were monitored by measuring time-dependent Ca(2+) responses in CHO-K1 cells transfected with a mu-opioid receptor and G(alpha 15) protein. The results indicate morphine to produce an initial increase in intracellular Ca(2+) concentration followed by a decrease below basal level. The occurrence of a sign-reversal was confirmed in native conditions of receptor-to-G protein coupling; the continuous in vivo infusion over a 2-week period of 0.31 mg rat(-1)day(-1) of fentanyl initially caused an increase of the mechanical threshold to induce a pain response (i.e. analgesia) that was followed by a decrease (i.e. hyperalgesia). The findings indicate that with opioid signaling systems, transduction mechanisms operate that may cause the sign of the effect to reverse not only when activation is discontinued but also whilst it is maintained at a constant magnitude.     

Zinc as a possible mediator of signal transduction in T lymphocytes

Our recent findings indicate that phorbol esters, the specific activators of protein kinase C induce the translocation of heavy metals (mostly: zinc) from the nucleus and mitochondria to the cytosol and microsomes of T lymphocytes. Phorbol ester treatment impairs the action of Ca-ionophores, this effect is mediated by intracellular heavy metal ions (most probably: by zinc). Zinc activates cytosolic protein kinase C, increases its affinity towards phorbol esters and contributes to its binding to plasma membranes. These results suggest that zinc may play a role in the "cross-talk" of second messengers and hence in signal transduction in T lymphocytes.     

Microfilament assembly modulates phospholipase C-mediated signal transduction by the TCR/CD3 in murine T helper lymphocytes.

Cytoskeletal involvement in the response to TCR/CD3 ligation and in signal transduction was investigated in a murine Th cell type 2 clone. Cells coated with the hamster anti-CD3 mAb, 145-2C11 (2C11 mAb), and exposed to goat anti-hamster demonstrated an increase in polymerized actin as well as an increase in inositol phospholipid hydrolysis mediated by activation of phospholipase C. Pretreatment with cytochalasins (Cyt) (D or B), drugs that interact with cellular actin, prevented actin polymerization, and augmented the initial rate and total amount of inositol phosphates produced. Drugs modifying microtubule function were ineffective. The intracellular Ca2+ rise attributed to InsP3 and InsP4 generated in response to CD3 perturbation was augmented by CytD. CytD treatment did not affect inositol phosphate generation resulting from the stimulation of guanine nucleotide-binding proteins with aluminium tetrafluoride, indicating that the action of CytD was specific for receptor-mediated inositol phospholipids. CytD decreased the rate of anti-CD3-induced receptor internalization. These data suggest that the assembly of microfilaments plays a role in CD3 internalization and that a CytD-sensitive mechanism uncouples the TCR/CD3 complex from phospholipase C-mediated signaling.     

Role of GM3-enriched microdomains in signal transduction regulation in T lymphocytes

Gangliosides, sialic acid containing glycosphigolipids, are ubiquitous constituents of cell plasma membranes. Each cell type shows a peculiar ganglioside expression pattern. In human T lymphocytes monosialoganglioside GM3 represents the main ganglioside constituent of cell plasma membrane where it is concentrated in glycosphingolipid-enriched microdomains (GEM). The presence of tyrosine kinase receptors, mono- (Ras, Rap) and heterotrimeric G proteins, Src-like tyrosine kinases (lck, lyn, fyn), PKC isozymes, glycosylphosphatidylinositol (GPI)-anchored proteins and, after T cell activation, the Syk-family kinase Zap-70, prompts these portions of the plasma membrane to be considered as “glycosignaling domains.” In particular, during T cell activation and/or other dynamic functions of the cell, such as apoptosis, key signaling molecules are recruited to these microdomains, where they strictly interact with GM3. The association of transducer proteins with GM3 in microdomains suggests that this ganglioside is the main marker of GEM in human lymphocytes and is a component of a cell plasma membrane multimolecular signaling complex involved in cell-cell interaction, signal transduction, and cell activation. Published in 2004. This revised version was published online in August 2006 with corrections to the Cover Date.     

Role of GM3-enriched microdomains in signal transduction regulation in T lymphocytes

Gangliosides, sialic acid containing glycosphigolipids, are ubiquitous constituents of cell plasma membranes. Each cell type shows a peculiar ganglioside expression pattern. In human T lymphocytes monosialoganglioside GM3 represents the main ganglioside constituent of cell plasma membrane where it is concentrated in glycosphingolipid-enriched microdomains (GEM). The presence of tyrosine kinase receptors, mono- (Ras, Rap) and heterotrimeric G proteins, Src-like tyrosine kinases (lck, lyn, fyn), PKC isozymes, glycosylphosphatidylinositol (GPI)-anchored proteins and, after T cell activation, the Syk-family kinase Zap-70, prompts these portions of the plasma membrane to be considered as "glycosignaling domains." In particular, during T cell activation and/or other dynamic functions of the cell, such as apoptosis, key signaling molecules are recruited to these microdomains, where they strictly interact with GM3. The association of transducer proteins with GM3 in microdomains suggests that this ganglioside is the main marker of GEM in human lymphocytes and is a component of a cell plasma membrane multimolecular signaling complex involved in cell-cell interaction, signal transduction, and cell activation.     

Role of cAMP-dependent signal transduction in the control of T lymphocyte activation

The role of cAMP in the regulation of antigen-dependent differentiation of T cells is discussed with consideration of the molecular mechanisms of cAMP effects. Characteristics of activation signal in various T lymphocyte subpopulations determining differential sensitivity to cAMP are reviewed. Specific attention is given to the involvement of the cAMP-dependent messenger system in the formation of the spectrum of secreted cytokines because their level and ratio determine the type of immune response.     

Specific activation of human T lymphocytes by Robinia pseudoacacia seeds' lectin.

Human peripheral blood and tonsil lymphocytes were fractionated into T and B cells by centrifugation after rosetting with native sheep erythrocytes and tested with Robinia pseudoacacia lectin. The purity of B- and T-enriched populations was checked by E-rosette formation or heterologous antisera specific for B or T lymphocytes. The proliferative response of T cells to Robinia lectin from all the donors tested was not found to differ from that of unfractionated cells, whereas no response of highly purified B cells could be observed to the lectin even with different concentrations of the lectin and different culture periods. B cells, however, were found to bind as much ³H labeled Robinia lectin as unfractionated lymphocytes. In addition, treatment of cells by antihuman T-lymphocyte antigen (HTLA) serum and complement before addition of Robinia lectin completely abolished their response, whereas similar treatment by antihuman B lymphocyte and monocyte antigen (HBLMA) serum did not prevent the T cells from incorporating thymidine. The Robinia lectin, like the other phytomitogens, thus appears to be a specific T-cell activator.     

Resonant activation of calcium signal transduction in neurons

The relevant parameters of calcium fluxes mediating activation of immediate-early genes and the collapse of growth cones in mouse DRG neurons in response to action potentials delivered in different temporal patterns were measured in a multicompartment cell culture preparation using digital flourescence videomicroscopy. Growth cone collapse was produced by trains of action potentials causing a large rise in [Ca²⁺]_i, but after chronic exposure to patterned stimulation growth cones regenerated and became insensitive to the stimulus-induced increase in [Ca²⁺]_i. Calcium reached similar peak concentrations, but the [Ca²⁺]_i increased more slowly than in naive growth cones (time constant of 6.0 s versus 1.4 s in naive growth cones). Semiquantitative PCR measurements of gene expression showed that pulsed stimulation delivered at 1-min intervals for 30 min induced expression of c-fos, but the same total number of action potentials delivered at 2-min intervals failed to induce c-fos expression, even though this stimulus induces a larger peak [Ca²⁺]_i than the effective stimulus pattern. The experiments suggest that the kinetics of calcium fluxes produced by different patterns of stimulation, and changes in the kinetics of calcium flux in neurons under different states of activation, are critical in determining the effects of action potentials on growth cone motility or expression of IE genes during development of neuronal circuits. We propose that differences in kinetics of individual reactions in the stimulus–response pathway may lead to resonance of activation in the neuron, such that certain processes will be selectively activated by particular temporal patterns of stimulation. 1994 John Wiley & Sons, Inc.     

IgD receptor-mediated signal transduction in T cells

Upregulation of immunoglobulin D-specific receptors (IgD-R) on CD4+ T cells may facilitate their interaction with specific carbohydrate moieties uniquely associated with membrane IgD on B cells. Previous studies have shown that upregulation of IgD-R facilitates cognate T-B cell interactions by mediating bidirectional signaling resulting in increased antibody responses and clonal expansion of antigen-specific T cells. Murine T hybridoma cells, 7C5, constitutively express IgD-R, as has been confirmed by staining with biotinylated IgD. Earlier studies have shown that inhibitors of protein tyrosine kinase (PTK) completely prevented upregulation of IgD-R in response to oligomeric IgD, suggesting that cross-linking of IgD-R may induce signal transduction and functional consequences through one or more PTK activation pathways, leading to upregulation of IgD-R. In the present study we show that cross-linking of IgD-R by oligomeric IgD indeed results in (a) T cell activation as seen by tyrosine phosphorylation of several intracellular proteins, (b) tyrosine phosphorylation of p56 Lck and PLC-gamma in 7C5 T hybridoma cells, and (c) phosphorylation of an approximately 29-kDa band that exhibits strong affinity for IgD. We analyzed tyrosine phosphorylation of p56 Lck and PLC-gamma in BALB/c splenic T cells that were exposed to oligomeric IgD both in vivo and in vitro. In vitro cross-linking as well as in vivo followed by in vitro cross-linking of IgD-R resulted in enhanced phosphorylation of p56 Lck and moderate tyrosine phosphorylation of PLC-gamma. These results suggest that interactions between IgD-R and IgD mediate signal transduction and support our previous findings that IgD-R+ T cells enhance cognate T cell-B cell interactions and antibody production.     

Ex vivo activation of tumor-draining lymph node T cells reverses defects in signal transduction molecules

 The adoptive transfer of tumor-draining lymph node (LN) T cells activated ex vivo with anti-CD3 and interleukin 2 (IL-2) mediates the regression of the poorly immunogenic murine melanoma D5. The efficacy of the activated LN cells is augmented when the sensitizing tumor is a genetically modified variant (designated D5G6) that secretes granulocyte/macrophage-colony-stimulating factor. In contrast to anti-CD3/IL-2-activated LN cells, adoptive transfer of freshly isolated tumor-draining LN T cells has no therapeutic activity. To determine whether the acquisition of antitumor function during ex vivo activation is associated with modifications in signal transduction capacity, the protein tyrosine kinases p56 ^lck and p59 ^fyn and proteins of the NF-κB family were analyzed in tumor-draining LN T cells. The levels of p56 ^lck and p59 ^fyn were lower in tumor-draining than in normal LN T cells and production of tyrosine-phosphorylated substrates was markedly depressed following anti-CD3 stimulation. After 5-day anti-CD3/IL-2 activation, levels of p56 ^lck and p59 ^fyn and protein tyrosine kinase activity increased. Interestingly, the levels of p56 ^lck , p59 ^fyn , and tyrosine kinase activity were higher in activated T cells derived from LN that drained D5G6 than they were in those from D5 tumors. In contrast, the cytoplasmic levels of c-Rel and Rel A were normal in freshly isolated tumor-draining LN, as was nuclear κB DNA-binding activity induced by anti-CD3 mAb or phorbol myristate acetate. Stimulation of activated LN cells with D5 tumor cells induced the nuclear translocation of NF-κB. These findings indicate that the recovery of proteins mediating signal transduction through the T cell receptor/CD3 complex in LN T cells activated ex vivo was associated with the acquisition of antitumor function. Received: 28 August 1997 / Accepted: 23 February 1998     

Block of T lymphocyte differentiation by activation of the cAMP-dependent signal transduction pathway.

Differentiation of T lymphocytes is a complex and finely tuned process. Here we show that treatment of mouse fetal thymus organ cultures with agents activating the cAMP-dependent signalling pathway results in the block of thymocyte differentiation. This is due to severe impairment of maturation beyond the CD4-/CD8- stage. In addition, rearrangements at the TCR alpha gene locus, but not at the TCR beta locus, are completely inhibited. The cAMP effect is reversible and is restricted to TCR alpha beta+ cells. cAMP acts both by triggering apoptosis and by inducing cell-cycle block in thymocytes. Thus, activation of the cAMP pathway provides a mechanism to modulate thymic function for hormones and ligands whose receptors are coupled to adenylate cyclase.     

FK-506 and cyclosporin A inhibit highly similar signal transduction pathways in human T lymphocytes.

This report compares the ability of cyclosporin A and FK-506 to inhibit human T cell activation triggered via cell surface molecules that utilize different intracellular processes. We stimulated highly purified peripheral blood T lymphocytes with mitogens (Con A and PHA), ionomycin + PMA, or monoclonal antibodies specific for cell surface antigens involved in activation (CD2, CD3, CD28) either in combination with each other or in conjunction with PMA. Using measurements of the proliferative response, IL-2 production, and changes in intracellular Ca2+ ([Ca2+]i), we demonstrate that FK-506 exerts its inhibitory effect on early events of T-cell activation in a manner indistinguishable from that of CsA. An important finding in this study is the strict correlation between those activation pathways that are inhibited by FK-506 and CsA and the requirement that the sensitive pathways induce a measurable rise in [Ca2+]i. This correlation held even for the CD28/CD2 pathway which was previously shown to be calcium-independent; however by employing FACS analysis of [Ca2+]i within individual cells, a subset of cells activated via CD28/CD2 was found to respond with a measurable rise in [Ca2+]i. We also noted that the proliferative response induced by certain stimuli, such as ionomycin + PMA and PHA + PMA, was partially resistant to FK-506 and CsA, while IL-2 production was completely suppressed. The partial FK-506/CsA-resistance of these responses was shown to be determined by the amount of PMA added to the cultures. We conclude from our investigations that FK-506 and CsA inhibit highly similar signal transduction pathways in human T lymphocytes.     

Cytoskeletal rearrangement during migration and activation of T lymphocytes.

T lymphocytes have an inherent ability to migrate along a chemotactic gradient, which enables them to exit the bloodstream and reach different tissues. Motile T cells display a polarized morphology with two distinct cell compartments: the leading edge and the uropod. During cell polarization, chemoattractant receptors, cell-adhesion molecules and cytoskeletal proteins are redistributed within these cellular compartments. The polarity of T lymphocytes changes during the establishment of antigen-specific cell-cell interactions, and this involves rearrangement of cytoskeletal proteins. This article discusses the regulation of these cytoskeletal rearrangements, and their role in the activation, migration and effector function of T cells.     

Transmembrane signaling during interleukin 1-dependent T cell activation. Interactions of signal 1- and signal 2-type mediators with the phosphoinositide-dependent signal transduction mechanism

The murine T lymphoma line, LBRM-33 1A5, requires synergistic signals delivered by phytohemagglutinin (PHA) and interleukin 1 (IL1) for activation to high level interleukin 2 production. The activation signals provided by PHA and IL1 were replaced by the Ca2+ ionophore, ionomycin, and the phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA), respectively. These observations supported a two-signal model for T cell activation involving increases in intracellular Ca2+ concentration ([Ca2+]i) (signal 1) and activation of protein kinase C (signal 2) as necessary and sufficient events. However, biochemical analyses revealed that additional signals were involved in the activation of LBRM-33 cells by both receptor-dependent and -independent mediators. Both signal 1-type mediators, PHA and ionomycin, exerted pleiotropic effects at the concentrations required for synergy with signal 2-type mediators (IL1, TPA). Within 1-2 min of addition, PHA stimulated phospholipid turnover, including hydrolysis of phosphatidylinositol 4,5-bisphosphate, Ca2+ mobilization, and protein kinase C activation. The [Ca2+]i increase induced by PHA was due to influx from both intracellular and extracellular Ca2+ pools. Similarly, ionomycin increased phospholipid turnover, [Ca2+]i, and directly affected protein kinase C activity in LBRM-33 cells. In contrast, the signal 2-type mediators, TPA and IL1, appeared to act by distinct intracellular mechanisms. TPA induced a protracted association of cellular protein kinase C with the plasma membrane, consistent with the two-signal activation model. Furthermore, acute TPA treatment inhibited PHA-stimulated inositol phosphate release and Ca2+ mobilization, suggesting that this mediator partially antagonized signal 1 delivery. IL1, in contrast, neither activated protein kinase C directly nor did it positively modulate the coupling of signal 1-type mediators to [Ca2+]i or protein kinase C via the phosphoinositide pathway. The intracellular signal delivered by IL1 is, therefore, generated through a mechanism distinct from or distal to the activation of protein kinase C. These studies indicate that the two-signal hypothesis, in its simplest form, is inadequate to explain the signals required for the initiation of IL1-dependent T cell activation.     

Effects of lectin activation on sialyltransferase activities in human lymphocytes

The effects of phytohemagglutinin (PHA) stimulation on the activities of sialyltransferase 1 (SAT-1), and sialyltransferase 3 (SAT-3), in human lymphocytes were investigated in vitro. For SAT-1 and SAT-3, respectively, the apparent Km values with variable CMP-NeuAc concentrations were 0.19 and 0.015 mM and with variable LacCer were 0.075 and 0.17 mM. Progressive increases in the activities of SAT-1 and SAT-3 were detected in lymphocytes stimulated with PHA, whereas no increase was observed in control lymphocytes incubated in culture medium alone. These increased activities occurred within 18-36 h of incubation and preceded optimum lymphocyte proliferation. Intact lymphocytes were needed for the lectin-stimulated increase of sialyltransferase activities because neither concanavalin A nor phytohemagglutinin added to the broken cell preparation modulated SAT-1 activity. The glycolipid products formed as a result of these enzymatic reactions in the presence of endogenous and exogenous acceptors were tentatively identified by thin-layer chromatography and autofluorography. The addition of exogenous LacCer to the SAT-1 assay resulted in the radiolabeling of a small amount of ganglioside GM1b (3.4%), but GM3 was the major labeled product (96%). When GgOse4Cer was added to the SAT-3 assay, 32% GM3 and 24.6% GM1b were detected while 44% consisted of glycolipids not labeled in assays performed without exogenous acceptors. Of the radioactivity transferred to endogenous acceptors, 81.3% was in GM3 and 14.6% in GM1b. These results demonstrate that the modulation of sialyltransferase activity occurs earlier than cellular activation.     

Defective T cell receptor-mediated signal transduction in memory CD4 T lymphocytes exposed to superantigen or anti-T cell receptor antibodies

Lymphocytes must promote protective immune responses while still maintaining self-tolerance. Stimulation through the T cell receptor (TCR) can lead to distinct responses in naive and memory CD4 T cells. Whereas peptide antigen stimulates both naive and memory T cells, soluble anti-CD3 antibodies and bacterial superantigens stimulate only naive T cells to proliferate and secrete cytokines. Further, superantigens, like staphylococcal enterotoxin B (SEB), cause memory T cells to become anergic while soluble anti-CD3 does not. In the present report, we show that signal transduction through the TCR is impaired in memory cells exposed to either anti-CD3 or SEB. A block in signaling leads to impaired activation of the kinase ZAP-70 so that downstream signals and cell proliferation do not occur. We further show that the signaling defect is unique to each agent. In anti-CD3-treated memory T cells, the src kinase Lck is only transiently activated and does not phosphorylate and activate ZAP-70. In SEB-treated memory T cells, ZAP-70 does not interact with the TCR/CD3 complex to become accessible to Lck. Finally, we provide evidence that alternative signaling pathways are initiated in SEB-treated memory cells. Altered signaling, indicated by an elevation in activity of the src kinase Fyn, may be responsible for memory cell anergy caused by SEB. Thus, differentiation of naive T cells into memory cells is accompanied by alterations in TCR-mediated signaling that can promote heightened recall immunity or specific tolerance.     

Receptor modulation and early signal transduction events in cytotoxic T lymphocytes inactivated by sensitive target cells.

In previous studies, we demonstrated that NK cells and lymphokine-activated killer cells were inactivated early in the lytic process by susceptible but not by resistant target cells (TC). We examined the functional status of human MHC-restricted CTL, after interaction with sensitive TC. Two CTL lines were generated in vitro by stimulation with irradiated PAMO, an EBV-transformed cell line. CTL were incubated for up to 4 h with an equal number of PAMO, then separated by a SRBC rosette assay. CTL lost greater than 60% of their lytic activity during the first 30 min of incubation, and greater than 90% by 4 h as assessed by their inability to lyse fresh TC. Inactivated CTL had 35% less serine esterase activity than did control CTL. IL-2 restored the lytic potential and serine esterase activity to normal values within 72 h. Exposure of CTL to PAMO for 4 h induced the modulation of 22 to 44% of TCR/CD3, CD4/CD8, and class I Ag from the cell surface. In contrast, the expression of CD69, and class II Ag increased and there was no change in the expression of CD2, CD28, or LFA-1 Ag. Furthermore, early metabolic events that usually follow CTL-ligand interaction such as phosphatidylinositol metabolism and transient increase in intracellular calcium, did not occur in inactivated CTL upon challenge with PAMO. PMA and the calcium ionophore A23187, restored cytolytic activity, indicating that protein kinase C can be activated and translocated in inactivated CTL. Our data suggest that TC-induced inactivation of CTL may be due to the modulation of key membrane molecules and the lack of certain secondary messengers involved in signal transduction.     

脂筏与T细胞信号转导

抗原提呈细胞将抗原加工处理后通过MHCⅠ/MHCⅡ类分子提呈供T细胞识别。TCR对抗原的识别引起一系列下游信号事件的发生,最终使T细胞激活,但对TCR复合物结合抗原后引起胞内区磷酸化的早期事件机制还不是很清楚。最近的研究揭示脂筏参与了这一早期信号事件的发生。脂筏是一种膜脂双层内含有的特殊微区,T细胞膜表面参与T细胞激活的各种关键信号分子都定位于脂筏。T细胞激活过程中脂筏通过聚集和重分配形成一个信号转导的平台。