Lymphocyte Signal Transduction Research Articles

Human alveolar macrophages inhibit receptor-mediated increases in intracellular calcium concentration in lymphocytes.

Prior studies have demonstrated that human alveolar macrophages (AM) are suppressive of lymphocyte function, through the mechanism of inhibition is unclear. In the current study, human AM inhibited receptor-mediated increases in intracellular calcium concentration ([Ca2+]i) in T cells, natural killer cells, and B cells. This effect was produced by either live or fixed AM, while peripheral blood monocytes caused a minimal reduction in [Ca2+]i. The inhibitory effect of AM was seen following 1 to 2 h of incubation with lymphocytes, was complete at 16 h, and did not affect ionomycin-mediated [Ca2+]i. Inhibition of [Ca2+]i by AM correlated with suppression of T-lymphocyte proliferation and cytotoxic T-lymphocyte activity in response to alloantigen and Staphylococcus A-induced immunoglobulin production. Our findings suggest that a membrane signal on AM is capable of inhibiting receptor-mediated signal transduction in lymphocytes and that this is likely a major mechanism by which immune responses are downregulated in the alveolus.

Suppression of Lymphocyte Signal Transduction by Murine Mastocytoma Ascites

The lymphocyte signal transduction, as determined by intracellular free Ca2+ mobilization of concanavalin A-stimulated T lymphocytes and of anti-immunoglobulin mu chain antibody-stimulated B lymphocytes, was suppressed in spleen cells from mice injected with murine P1.HTR mastocytoma-induced ascites and in spleen cells treated with the ascites in vitro. The suppression was observed both at the peak level and in the reactive pattern of Ca2+ influx. In the suppression, the ascites were replaceable with tumor culture supernatants or tumor homogenates. Correspondingly, primary and secondary cytotoxic T lymphocyte (CTL) responses of DBA/2 mice to allogeneic antigen were also significantly suppressed by injection of the syngeneic P1.HTR tumor-derived ascites. This new finding suggested that the mechanism of the tumorous ascites or of the tumor-derived factor-mediated immunosuppression involves at least in part the suppression of the early event of the signal transduction for lymphocyte activation.

AGE-RELATED DIFFERENCES IN THE EFFECTS OF CYCLOSPORINE ON LYMPHOCYTE INTRACELLULAR FREE CALCIUM

The effect of cyclosporine on lymphocyte intracellular free calcium [( Ca2+]i) is controversial, and potential age-related differences in lymphocyte CsA sensitivity have not been studied. We measured the mitogen-induced change in [Ca2+]i in peripheral blood lymphocytes (PBLs) following intravenous CsA infusion (5 mg/kg) in neonatal pigs and found a significantly reduced calcium response compared with control (P = 0.02). This was associated with an elevation in resting [Ca2+]i in the neonatal PBLs 24 hr following the CsA infusion (P = 0.02). These changes in lymphocyte [Ca2+]i were associated with suppression of cell proliferation. Neonatal PBLs in mixed lymphocyte cultures showed a greater PHA-induced change in [Ca2+]i (delta[Ca2+]i) compared with mature PBLs (P = 0.0007). The addition of CsA (1 microgram/ml) to mitogenic- and allogeneic-stimulated cultures did not affect resting [Ca2+]i or delta[Ca2+]i in either neonatal or mature PBLs. Our results demonstrate significant differences in calcium responses in neonatal lymphocytes following CsA infusion and allogeneic stimulation. This implies that there are age-related differences in CsA effects at or proximal to the level of calcium release and/or sequestration in the lymphocyte signal transduction pathway, and that elevated resting intracellular calcium levels may be indicative of reduced responsiveness, possibly through feedback inhibition of tyrosine kinase activity.

Interaction of CD2 with its ligand lymphocyte function-associated antigen-3 induces adenosine 3',5'-cyclic monophosphate production in T lymphocytes.

CD2 (T11, the T cell E receptor), a nonpolymorphic 47- to 55-kDa glycoprotein, is a T cell-specific surface protein that plays an important role in T lymphocyte adhesion, signal transduction, and differentiation. A natural ligand of CD2 is lymphocyte function associated Ag-3 (LFA-3 (CD58)), a widely expressed glycoprotein of 50 to 70 kDa. The physiologic interaction of CD2 with LFA-3 functions to increase intercellular adhesion and plays a role in T cell activation. This interaction, however, in the absence of other stimuli, has not previously been shown to induce intracellular signals such as Ca2+ mobilization or IL-2 production. To investigate whether cAMP may play a role in ligand-triggered CD2-mediated signal transduction, we have studied the ability of purified LFA-3 and anti-CD2 mAb to induce changes in intracellular cAMP content in murine Ag-specific T cell hybridomas that stably express wild-type and mutated human CD2 molecules. By using a RIA sensitive to the femtomolar range and specific for cAMP, we demonstrate that purified LFA-3, like anti-CD2 mAb, is capable of inducing marked, transient increases in the intracellular concentration of cAMP. Presentation of purified LFA-3, like anti-CD2 mAb, is capable of inducing marked, transient increases in the intracellular concentration of cAMP. Presentation of purified LFA-3 alone to CD2-expressing hybridoma cells, however, did not stimulate phosphatidylinositol turnover nor IL-2 production. The cytoplasmic domain of CD2 is necessary for these ligand-induced cAMP changes, demonstrating that LFA-3 binding to CD2 transduces a signal to the cell. Experiments using the phosphodiesterase inhibitor 3-isobutyl-1-methyl-xanthine showed that CD2-mediated regulation of cAMP levels occurs primarily by the stimulation of cAMP production rather than by the inhibition of cAMP degradation. These results demonstrate that the interaction of LFA-3 with CD2, in the absence of other stimuli, is capable of initiating intracellular biochemical changes and suggest that CD2/LFA-3 interactions may regulate T cell function at least in part through the generation of intracellular cAMP.

Is cyclophilin involved in the immunosuppressive and nephrotoxic mechanism of action of cyclosporin A?

In this report we have approached two questions relating to the mechanism of action of cyclosporin A (CsA). First, we address whether the major cytosolic protein for CsA, cyclophilin, is directly involved in mediating the immunosuppressive activity of this drug, and, in particular, whether inhibition of this protein's peptidyl-prolyl cis-trans isomerase (PPIase) activity results in inhibition of murine T cell activation. Second, we ask whether the nephrotoxicity observed with CsA is related to inhibition of PPIase-dependent pathways in cells other than lymphocytes. Using a series of 61 cyclosporin analogues, we generally found a good correlation between cyclophilin binding and immunosuppressive activity for the majority of analogues analyzed. However, a number of compounds of distinct structural classes were found that could interact with cyclophilin but were much less immunosuppressive than expected. The inability of these analogues to inhibit lymphocyte activation could not be explained by their failure to enter the cell and bind to cyclophilin under the conditions used in the cellular assays. Surprisingly, a nonimmunosuppressive analogue, MeAla-6, which bound well to cyclophilin and was active as a PPIase inhibitor, did not induce renal pathology in vivo. Furthermore, another analogue, MeBm2t, which was immunosuppressive in vitro, possessed little or no activity as a PPIase inhibitor. These findings pose serious questions concerning a direct role of cyclosporin in mediating CsA's immunosuppressive and nephrotoxic activities. In addition, they raise doubts about whether PPIase has a direct function in lymphocyte signal transduction.

Observations on the mechanism of action of FK-506: A pharmacologic probe of lymphocyte signal transduction

Le FK-506, immunodepresseur produit par un streptomyces, est connu pour inhiber la proliferation des lymphocytes et la secretion des lymphokines. L'action s'exerce au niveau biochimique sur la proline isomerase, mais une inhibition d'oncogenes comme c-myc, a ete aussi observee. La mise en evidence des sites d'intervention de FK-506 dans la transduction mitotique du lymphocyte, devrait permettre d'optimiser les proprietes immunosuppressives de cette molecule

Ion Channels and Signal Transduction in Lymphocytes

T and B lymphocytes play fundamental and diverse roles in the immune response, which include recognition of antigens, secretion of Iymphokines and antibodies, and killing of foreign or virus-infected cells. Ionic fluxes associated with lymphocyte activation, mitogenesis, and volume regulation have been studied extensively in cell suspensions (for reviews, see 67, 103, 124, Grinstein & Foskett, this volume). More recently, the advent of patch­ clamp recording techniques (55) and video imaging of fluorescent ion­ sensitive dyes (53, 131) has placed a new emphasis on the responses of individual cells in the immune system. In this review we address recent advances in the exploration of ion-channel functions in T and B lymphocytes at the single-cell level, with particular emphasis on three lymphocyte be­ haviors: cell activation and mitogenesis, cytotoxicity, and volume regulation.

Involvement of the PPPGHR motif in T cell activation via CD2.

Prior studies identified a segment of the CD2 cytoplasmic domain between amino acid (aa) residues 253 and 287 as important in T lymphocyte signal transduction. This region contains two repeats of the sequence motif PPPGHR, thought to form a "cage" structure involved in CD2-mediated signaling. To evaluate this segment, a series of mutant human CD2 molecules were produced by oligonucleotide-directed mutagenesis and inserted into the ovalbumin-specific, I-Ad-restricted murine T-T hybridoma 3DO54.8 using the DOL retroviral system. CD2 M1 (271-272), CD2 M2 (278-279), and CD2 M4 (264-265) mutants replaced the positively charged adjacent aa histidine and arginine (HR) in the wild-type CD2 sequence with aspartic and glutamic acid (DE) at positions 271-272, 278-279, and 264-265, respectively. In addition, a truncation mutant, CD2 M3 (268), containing only 57 of the 117 cytoplasmic aa and terminating before the second PPPGHR sequence, was generated. Stimulation of transfectants CD2 FL, CD2 M1 (271-272), and CD2 M2 (278-279) with anti-T11(2) + anti-T11(3) antibodies resulted in a rise in cytosolic-free calcium [( Ca2+]i) and subsequent interleukin 2 (IL-2) secretion. In contrast, CD2 M4 (264-265) transfectants could not be activated in either assay. Thus, alteration of histidine 264 and/or arginine 265 within the first PPPGHR motif affects the process of signal transduction via CD2, whereas identical mutations in residues at 271-272 or 278-279 were individually without effect. Consistent with these data, CD2 M3 (268) transfectants were able to generate a detectable amount of IL-2 via CD2 triggering. These data support the notion that the PPPGHR motif at aa 260-265 is important for activation of T lymphocytes via the CD2 molecule.

Early effects of the immunosuppressive drug FK-506 on signal transduction in lymphocytes

Early effects of the immunosuppressive drug FK-506 on signal transduction in lymphocytes

Inositol 1,4,5-trisphosphate- and arachidonic acid-induced calcium mobilization in T and B lymphocytes

Inositol triphosphate (IP3) formation and increase in intracytoplasmic calcium are mediators of signal transduction in lymphocytes. It has been proposed that IP3 induces Ca 2+ release from intracellular stores. It is in order to study the relationship between these two events that we have analyzed the effect of IP3 addition on Ca 2+ mobilization in permeabilized resting T and B lymphocytes, EBV-B lymphocytes, and HTLV1-T lymphocytes. IP3 induces a rapid and significant release of Ca 2+ from the endoplasmic reticulum in a dose-dependent manner. Ca 2+ release is more sensitive to IP3 addition in cycling cells (EBV-B lymphocytes and HTLV1-T lymphocytes) than in resting T and B lymphocytes. Arachidonic acid (AA) induces Ca 2+ release from the endoplasmic reticulum (ER) in a manner similar to that of IP3. Neither component has an effect on Ca 2+ accumulated in mitochondria, and they have no additive effects suggesting that they act on a similar Ca 2+ pool. These results directly demonstrate that in T and B human lymphocytes IP3 mobilizes Ca 2+ from ER as in other cellular systems and that other potential second messengers, namely AA, could play a significant role in the internal mobilization of calcium during T and B lymphocyte activation.

Partial deletions of the cytoplasmic domain of CD2 result in a partial defect in signal transduction.

CD2 (T11, the T cell erythrocyte receptor or the SRBC receptor), a nonpolymorphic 47- to 55-kDa glycoprotein, appears to play a role in T lymphocyte adhesion, signal transduction, and differentiation. Pairs of anti-CD2 mAb induce T cell proliferation, suggesting that CD2 may be an Ag-independent pathway of T cell activation. We have expressed the human CD2 and a number of cytoplasmic domain deletion mutants of CD2 in an Ag-reactive murine hybridoma. We have previously shown that a cytoplasmic domain deletion mutant, CD2 delta B, in which the carboxyl-terminal 100 amino acids have been deleted, is no longer capable of signaling through CD2. Here we have expressed a second cytoplasmic domain deletion mutant, CD2 delta S, in which the terminal 41 amino acids have been removed, including the region with greatest conservation between the mouse, rat, and human species. CD2 delta S+ hybridomas were able to respond to Ag and to LFA-3 plus an anti-CD2 mAb. Although the CD2 delta S+ hybridomas responded comparably to the wild-type CD2+ hybridomas to certain pairs of anti-CD2 mAb (e.g., MT110 + 9-1 mAb), these CD2 delta S+ hybridomas were markedly deficient in their ability to respond to other pairs of stimulatory anti-CD2 mAb (e.g., 9.6 + 9-1 mAb). These data suggest that the cytoplasmic domain may have several functional regions, as partial deletions of the cytoplasmic domain appear to result in partial defects in signal transduction.

Ion Channels And Signal Transduction In Lymphocytes

Macrophage polarization refers to how macrophages have been activated at a given point in space and time. Polarization is not fixed, as macrophages are sufficiently plastic to integrate multiple signals, such as those from microbes, damaged tissues, and ...Read More

Effects of cadmium on lymphocyte activation

The effects of cadmium (Cd) on phytohemoagglutinin or phorbol myristate acetate-induced lymphocyte activation were investigated and a dose-dependent inhibition of cell proliferation was found. Kinetic studies revealed that the Cd-sensitive step is an early event of T cell stimulation. Failure of IL2 secretion and reduction of IL2 receptor expression in the Cd-treated cells are also reported. Regardless of which mechanism is responsible for Cd effects, our studies show that the inhibition of lymphocyte activation is associated with reduced [ 3H]phorbol dibutyrate binding to Ca 2+-phospholipid-dependent protein kinase and altered breakdown of phosphatidylinositols. Thus, Cd interferes with two biochemical events which play a critical role in lymphocyte signal transduction and activation.

T3 glycoprotein is functional although structurally distinct on human T-cell receptor gamma T lymphocytes.

The T-cell receptor (TCR) gamma gene product occurs in association with T3 (CD3) polypeptides on the surface of human T lymphocytes. TCR gamma lymphocytes express arrays of T3 polypeptides distinct from those typically observed on TCR alpha beta lymphocytes. This report demonstrates that identical T3 gamma, delta, and epsilon polypeptides are synthesized by TCR gamma lymphocytes and TCR alpha beta lymphocytes. However, the processing of T3 delta oligosaccharides is distinct in the two cell types. This observation may suggest distinct quaternary structures of these receptor complexes. Despite these structural differences, the T3 molecule on TCR gamma lymphocytes is functional. It is associated with and comodulates with TCR gamma and it serves as a substrate for protein kinase C-mediated phosphorylation. Anti-T3 monoclonal antibodies induce a rapid increase in cytoplasmic free calcium, indicating that the receptor complex is involved in signal transduction and triggering of TCR gamma lymphocytes.

Tyrosine protein kinases and their substrates in human leukemia cells

We have quantitated tyrosine protein kinase (TPK) activity in particulate and cytosolic fractions from human leukemia cells. Slowly proliferating cells from patients with chronic lymphocytic leukemia (CLL) had levels of TPK similar to those of quiescent normal lymphocytes. Cells from patients with acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML) and chronic granulocytic leukemia (CGL) contained markedly lower levels of TPK activity, similar to the levels in phytohaemagglutinin-stimulated (proliferating) normal lymphocytes and in bone marrow cells. This suggested that TPK is part of a mechanism for transducing growth signals and is down-regulated following signal transmission. We also identified endogenous substrates for TPK in leukemic cells. Particulate fractions from ALL, CLL and AML cells contained substrates identical to those previously detected in normal lymphocytes. In particular, a 38kD substrate thought to be involved in early stages of growth signal transduction in normal lymphocytes was found in all samples of these groups examined. Cytosolic fractions from these groups of leukemia cells contained higher molecular weight substrates not found in resting or proliferating normal lymphocytes or bone marrow cells. In contrast, TPK substrates in both particulate and cytosolic fractions from CGL cells resembled those of normal bone marrow cells in that only proteins with molecular weight below 40kD were labelled on tyrosine. We conclude that leukemic cells do not contain higher levels of TPK than do normal hemopoietic cells. Qualitative differences in TPK species or in their substrates may result in aberrant regulation of proliferation in leukemic cells. However, we cannot exclude the possibility that additional TPK substrates detected in leukemic cells were a feature of the normal equivalent hematopoietic cells from which the leukemia cells were derived.