Biomedical subjects

M Jondal

Publications and source records attributed to M Jondal.

At least 73 records · Page 4Linked to original sources

PubMed1992

Potentiation of transmembrane signaling by cross-linking of antibodies against the beta chain of the T cell antigen receptor of JURKAT T cells.

Three monoclonal antibodies (mAb) 2D1, 3B9, and 3B12 were produced by immunizing BALB/c mice with JURKAT cells. These mAb induce comodulation of the TCR/CD3 complex expressed on JURKAT cells, but do not react with the CD3- JURKAT variant, J.RT3.T3.1. Immunoprecipitation studies with detergent-solubilized JURKAT cell lystes indicate that these mAb react with proteins having characteristics of the TCR molecules. Their low reactivity with peripheral blood mononuclear cells (PBMC) and lack of reactivity with other CD3+ T cell lines suggest that they may be anti-idiotypic mAb. Results from binding inhibition assays, reactivity with PBMC, and generation of transmembrane signals suggest that these three anti-TCR mAb recognized different epitopes on the TCR beta chain of JURKAT cells. Although the three mAb are capable of inducing the production of inositol phosphates and cytosolic free Ca2+ increase in JURKAT cells, their stimulatory capacities vary and are lower than that observed by anti-CD3 antibody (OKT3) stimulation. However, crosslinking these mAb with rabbit antimouse immunoglobulins potentiates the stimulatory response to comparable levels induced by OKT3. These mAb could be useful as tools to study V beta 8+ T cells in relation to antigen-specific activation.

Animals↗

PubMed1992

In vivo priming of cytotoxic T lymphocyte responses in relation to in vitro up-regulation of major histocompatibility complex class I molecules by short synthetic peptides.

Cytotoxic T lymphocytes (CTL) recognize target antigens as short peptides presented by major histocompatibility complex class I molecules (MHC-I). Externally added peptides can sensitize target cells by binding directly to MHC-I without any need for internal processing. Those which are similar in length to endogenously processed peptides are more potent in this respect than slightly longer peptides. Peptide MHC-I interactions can also be reflected as up-regulation of MHC-I in vitro on certain cells. We have compared the capacity of Db, Kb- and Ld-binding peptides, which are slightly different in length, to up-regulate MHC-I in vitro with their immunogenicity in vivo, in relation to generation of CTL responses. A clear correlation between these two different functions was found. We have also modified a 9-mer Db-binding peptide by adding cystein to the amino terminus and lysine to the amino- or carboxy terminus and studied the effects on MHC-I up-regulation and in vivo immunogenicity. Cystein and lysine contain reactive groups which are likely to influence the binding of modified peptides into the antigen-binding groove of Db. These small modifications of the optimal 9-mer peptide strongly influenced their functions but still there was a correlation between MHC-I up-regulation and CTL responses. Up-regulation of MHC-I in vitro may reflect a capacity of peptides to accumulate on the surface of particular antigen-presenting cells in vivo.

Amino Acid Sequence↗

PubMed1992

Mitogen stimulation of T-cells increases c-Fos and c-Jun protein levels, AP-1 binding and AP-1 transcriptional activity.

We have analysed the effect of mitogenic lectins on c-Fos and c-Jun protein levels as well as on activator protein-1 (AP-1) binding and enhancer activity in Jurkat T-cells. Both c-Fos and c-Jun protein levels were increased after Con A and PHA stimulation. Since T-cell stimulation increases both intracellular Ca2+ and cAMP levels and activates protein kinase C (PKC), the possible involvement of these intracellular messengers in c-Fos and c-Jun induction was tested. PMA, which directly activates PKC, mimicked the effect of the lectins on c-Fos and c-Jun, but elevation of either intracellular Ca2+ or cAMP levels had little or no effect. The mitogen-induced increase of c-Fos and c-Jun immunoreactivity was inhibited by H-7, a kinase inhibitor with relatively high specificity for PKC, and less efficiently by H-8, a structurally related kinase inhibitor less active on PKC, but more active on cyclic nucleotide-dependent kinases. Con A stimulation was found to increase both binding of AP-1 to the AP-1 consensus sequence, TRE, and AP-1 enhancer activity, in Jurkat cells. PMA was also found to increase the AP-1 enhancer activity, whereas elevation of Ca2+ or cAMP had only minor effects. We conclude that stimulation with mitogenic lectins is sufficient to increase both c-Fos and c-Jun protein levels, AP-1 binding and AP-1 enhancer activity in Jurkat cells and that they act via mechanisms that could involve the activation of PKC.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1992

Cellular signaling in thymocyte apoptosis.

Induction of apoptosis (programmed cell death) in response to T cell receptor triggering is now thought to be involved in the process of negative selection in the thymus, and current work is therefore aimed at investigating how apoptosis is regulated within the cells. To this end, recent work has implicated several of the well-known signal transduction pathways already known to regulate T cell activation in the regulation of apoptosis in thymocytes. In particular, elevations of the cytosolic Ca2+ level or increases in cAMP can trigger thymocyte apoptosis, whereas activation of protein kinase C appears to inhibit apoptosis in response to either induction pathway. Moreover, crosslinking of Thy-1, CD4, or CD8 leads to potentiation of T cell receptor-mediated cell death, effects that appear to involve protein tyrosine kinase activation. These observations may be relevant to the question of how T cell receptor occupancy can mediate both differentiation and death during intrathymic T cell development.

Animals↗

PubMed1991

CD3-dependent increase in cyclic AMP in human T-cells following stimulation of the CD2 receptor.

We have previously shown that stimulation of the Ti/CD3 receptor complex on human T-cells potentiates adenylate cyclase activation by adenosine or forskolin. Anti-CD2 receptor antibodies shared with anti-CD3 antibodies the ability to potentiate dose dependently the adenosine- and forskolin-stimulated cyclic adenosine monophosphate (cAMP) accumulation, whereas stimulation of the CD45 receptor had no effect on cyclase activity. Modulation of the CD3 complex with anti-CD3 antibodies was found to decrease the CD2 receptor effect on adenylate cyclase activity greatly. The possible involvement of CD3-stimulated phospholipase C (PLC) activation on the cAMP potentiation was examined using HPB-ALL cells that express a CD3 complex with a defect coupling to PLC. Stimulation of the CD3 complex on HPB-ALL cells had only slight effects on adenosine-stimulated cAMP formation, whereas the effect on forskolin-stimulated cAMP was virtually unchanged. The CD3 effect was further analyzed in Jurkat cell membranes. In contrast to the results obtained after stimulation of intact cells, it was found that OKT3 stimulation of membranes did not potentiate the forskolin response. Finally, we tested whether inhibition of endogenous adenylate cyclase agonist production affected the CD3 effect. Inhibition of adenosine production or adenosine breakdown with 8-p-sulphophenyl theophylline (8-PST) or adenosine deaminase (ADA), respectively, did not alter the CD3 effects. Indometacin, which inhibits prostaglandin production, also had no effect. Together, these data show that stimulation of the CD2 receptor potentiates adenylate cyclase responses by a mechanism that is dependent on CD3 expression. Furthermore, the CD3 effect on cAMP appears to be mediated by two different mechanisms, one which is, and one which is not dependent on PLC. Finally, this effect is not due to an endogenous production of adenylate cyclase agonists.

Adenosine↗

PubMed1991

Translocation of the alpha- and beta-isoforms of protein kinase C following activation of human T-lymphocytes.

We have analyzed how activation of human Jurkat T-cells by the mitogenic lectin, concanavalin A (Con A), may affect the cellular distribution of the alpha- and beta-isoforms of protein kinase C (PKC) in T-cells. In non-stimulated cells almost all of the alpha- and beta-PKC was localized to the cytoplasmic compartment. Stimulation with Con A caused a transient translocation of both alpha- and beta-PKC from the cytoplasm to the cell membrane. The alpha-isoform appeared to be translocated to a somewhat greater extent and for a longer period of time than the beta-form. Translocation was maximal between 1 and 5 min for both of the isoforms. 30 min after stimulation, beta-PKC had returned to basal levels, whereas a substantial amount of alpha-PKC remained associated with the particulate fraction. We conclude that activation of human T-cells causes the translocation of at least two different isoforms of PKC, alpha-PKC and beta-PKC.

Amino Acid Sequence↗

PubMed1991

Modification of membrane phospholipid fatty acyl composition in a leukemic T cell line: effects on receptor mediated intracellular Ca2+ increase.

The effect of modifying fatty acyl composition of cellular membrane phospholipids on receptor-mediated intracellular free Ca2+ concentration ([Ca2+]i) increase was investigated in a leukemic T cell line (JURKAT). After growing for 72 h in medium supplemented with unsaturated fatty acids (UFAs) and alpha-tocopherol, the fatty acyl composition of membrane phospholipids in JURKAT cells was extensively modified. Each respective fatty acid supplemented in the culture medium was readily incorporated into phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine and phosphatidylcholine in the JURKAT cells. The total n-6 fatty acyl content was markedly reduced in phosphatidylinositol and phosphatidylcholine of cells grown in the presence of n-3 fatty acids (alpha-linolenic acid, eicosapentaenoic acid and docosahexaenoic acid). Conversely, in the presence of n-6 fatty acids (linoleic acid and arachidonic acid), the total n-3 fatty acyl content was reduced in all the phospholipids examined. In n-3 and n-6 polyunsaturated fatty acid (PUFA) modified JURKAT cells, the total n-9 monounsaturated fatty acyl content in the phospholipids were markedly reduced. Changing the fatty acyl composition of membrane phospholipids in the JURKAT cells appears to have no affect on the presentation of the T cell receptor/CD3 complex or the binding of anti-CD3 antibodies (OKT3) to the CD3 complex. However, the peak increase in [Ca2+]i and the prolonged sustained phase elicited by OKT3 activation were suppressed in n-3 and n-6 PUFA but not in n-9 monounsaturated fatty acid modified cells. In Ca2+ free medium, OKT3-induced transient increase in [Ca2+]i representing Ca2+ release from the inositol 1,4,5-trisphosphate-sensitive Ca2+ stores, were similar in control and UFA modified cells. Using Mn2+ entry as an index of plasma membrane Ca2+ permeability, the rate of fura-2 fluorescence quenching as a result of Mn2+ influx stimulated by OKT3 in n-9 monounsaturated fatty acid modified cells was similar to control cells, but the rates in n-3 and n-6 PUFA modified cells were significantly lower. These results suggest that receptor-mediated Ca2+ influx in JURKAT cells is sensitive to changes in the fatty acyl composition of membrane phospholipids and monounsaturated fatty acids appears to be important for the maintenance of a functional Ca2+ influx mechanism.

Antigens, Differentiation, T-Lymphocyte↗

PubMed1991

Induction of DNA fragmentation in chronic B-lymphocytic leukemia cells.

Chronic lymphocytic leukemia of B cell type (B-CLL) is a neoplastic disorder characterized by the accumulation of small resting lymphocytes in the periphery. The phenotype of these cells suggests that they are "frozen" at an early stage of maturation. Glucocorticoid hormones are commonly used to treat patients with B-CLL, resulting in a reduction in the peripheral lymphocyte count by an undefined mechanism. Here we report that glucocorticoids stimulate DNA fragmentation characteristic of a suicide process known as apoptosis or programmed cell death (PCD) in suspensions of cells from patients with B-CLL. The effects can be mimicked by Ca2+ ionophore and involve a sustained increase in the cytosolic Ca2+ concentration. Specific antibodies binding to membrane-associated IgM on the leukemic cells can also induce PCD by a similar mechanism. Phorbol esters block DNA fragmentation and cell killing in response to all of the agents, suggesting that activation of protein kinase C desensitizes the cells to PCD. Targeting the B-CLL cells with antibodies that induce an unbalanced, sustained Ca2+ increase may therefore represent a rational strategy for the destruction of leukemic cells.

Antibodies, Monoclonal↗

PubMed1991

Phospholipase C treatment of certain human target cells reduces their susceptibility to NK lysis without affecting binding or sensitivity to lytic granules.

Phosphatidylinositol-specific phospholipase C (PI-PLC) is an enzyme that has the capacity to release glycosyl-phosphatidyl inositol (G-PI)-anchored proteins from the cells surface. Pretreatment of the human T-cell leukemia cell line Molt-4 with PI-PLC resulted in a decrease in the susceptibility to lysis by natural killer (NK) cells. Treatment of the erythroleukemia cell line K562 with PI-PLC had no effect on its NK susceptibility. PI-PLC-treated and untreated Molt-4 bound equally well to lymphocytes in target-binding studies with effector cell preparations enriched for NK cells. Susceptibility to cytolytic granules isolated from rat LGL tumor cells remained the same after treatment of Molt-4 or K562 with PI-PLC. Combined treatment of Molt-4 with PI-PLC and rlFN-alpha or rlFN-gamma resulted in additive reductions of the NK susceptibility, suggesting that PI-PLC and interferons act on different mechanisms to protect cells from NK lysis. When expression of a number of antigens on Molt-4 and K562 was analyzed in flow cytometry, only the expression of CD58 was reduced after PI-PLC treatment. The susceptibility of Con A blasts to MLR derived cytotoxic T-cells was not altered by treatment with phospholipase. These data suggest that PI-PLC treatment reduces the capacity of some target cells to activate NK cells upon contract. The mechanism behind this phenomenon is presently unclear.

Antigens, CD↗

PubMed1991

Monoclonal antibodies to conserved regions of the major core protein (gag24) of HIV-1 and HIV-2.

Five mouse monoclonal antibodies were raised against a recombinant protein comprising the complete sequence of gag24 protein from HTLV-IIIB. All monoclonal antibodies recognized the native protein in enzyme-linked immunosorbant assay (ELISA) and Western blots. All monoclonal antibodies also cross-reacted with an human immunodeficiency virus type 2 (HIV-2) strain in western blots, whereas only one antibody detected HIV-2 p25 in ELISA. By using synthetic peptides, cross-reacting epitopes were mapped and three regions were defined. The conserved immunogenic sites were located in the carboxyterminal region of the protein. Inhibition experiments with human sera showed that this region is also immunogenic in humans.

Animals↗

PubMed1991

SPAM-8, a mouse-human heteromyeloma fusion partner in the production of human monoclonal antibodies. Establishment of a human monoclonal antibody against cytomegalovirus.

A heteromyeloma (mouse x human) cell line (SPAM-8) was produced by fusing mouse myeloma cells (SP2/0) with human peripheral blood lymphocytes. The cells were sensitive to aminopterin and resistant to ouabain. The cells showed a doubling time of about 19 hours and a cloning efficiency of 0.8 cells/well (to obtain growth in 50% of wells seeded) using mouse thymocytes as feeder cells. The number of chromosomes was about 86 and 1% of the total DNA was of human origin. Fusion of SPAM-8 cells with lymphocytes prepared from human spleens resulted in approximately one hybridoma per 10(5) seeded lymphocytes. A trioma (human x [mouse x human]) cell line was established by fusing cells of an Epstein-Barr virus-transformed B cell line with SPAM-8 cells. The trioma cells produced antibodies (IgG1, K) against cytomegalovirus, in a concentration of 7 micrograms/ml in spent medium, over a period of six months of continuous culture. The results obtained indicate that the heteromyeloma SPAM-8 may be used as a fusion partner in the production of human monoclonal antibodies.

Animals↗

PubMed1991

Toxic effects of interleukin-2-activated lymphocytes on vascular endothelial cells.

IL-2-activated lymphocytes (LAK cells) show increased adherence to, and killing of, human vascular endothelial cells compared to resting lymphocytes. In the present work, we have found that supernatants from LAK cell cultures also are toxic to human umbilical vein endothelial cells (HUVEC) when tested for 48 h in a neutral red uptake assay. Recombinant TNF-alpha and IFN-gamma at high concentrations are also toxic under the same test conditions, and TNF-alpha was directly detected in LAK cell supernatants. An inconsistent inhibition of toxicity was found with anti-TNF-alpha whereas anti IFN-gamma antibodies had a partial inhibitory effect. The susceptibility of HUVEC to cellular killing by LAK cells could be up- and down-regulated with insulin-like growth factor I and IFN-gamma, respectively. It is concluded that damage to vascular endothelium during high dose IL-2 treatments may be partially related to an excessive production of lymphokines such as IFN-gamma and TNF-alpha. IFN-gamma may, in addition, be protective for HUVEC during cellular interactions with LAK cells.

Culture Media↗

PubMed1991

Identification of amino acids in the V3 region of gp120 critical for virus neutralization by human HIV-1-specific antibodies.

The importance of the dependence on single amino acids in the V3 region of HIV-1 gp120 was evaluated for virus neutralization and antibody-dependent cellular cytotoxicity (ADCC). Synthetic overlapping 15-mer peptides and a set of omission peptides covering amino acids 301-317 were used. Sera from 29 HIV-1-infected individuals at different stages of disease were tested for neutralization, ADCC and specific IgG reactivity. Six HIV-1 neutralizing monoclonal antibodies (mAb) acted as controls. All mAb reacted with a region (amino acids 304-318) of gp120, previously shown to induce neutralizing antibodies. The amino acids essential for reactivity were identified to be within the sequence GPGR (amino acids 312-315). The importance of this region for occurrence of neutralizing antibodies in infected humans was investigated using the same set of peptides. Out of 29 individuals, 21 were found to have neutralizing antibodies in titres between 100 and 1000. Among the neutralization-positive sera, 17/21 (81%) reacted with amino acids 304-318, compared with only one of eight sera (13%) negative in neutralization. When any of the four amino acids G, P, G or R were deleted, the seroreactivity decreased considerably. The conserved sequence GPGR was therefore considered to be the most important for neutralization in this region in human sera as well. Thus, the conserved sequence GPGR in the V3 region of gp120 is critical for virus neutralization by human HIV-1-specific antibodies.

Acquired Immunodeficiency Syndrome↗

PubMed1991

Expression of interleukin-1 alpha, interleukin-1 beta and interleukin-6 in chronic B lymphocytic leukaemia (B-CLL) cells from patients at different stages of disease progression.

We have previously found that isolated B-CLL cells from progressive disease produce less interleukin-1 beta (IL-1 beta) as compared with cells from patients with indolent disease. Here we extend that finding to include measurements of IL-1 beta mRNA and secretion of IL-1 alpha and interleukin-6 (IL-6). As before, a lower production of IL-1 beta was found in cells from progressive disease. IL-6 was produced by cells from patients at all stages, with a tendency to follow the IL-1 beta production. Low secretion of IL-1 alpha was noted. When viable cells were permeabilized and analysed at the single cell level with monoclonal antibodies, most B-CLL cells were found to contain IL-1 alpha. A minor fraction of non-permeabilized cells expressed IL-1 alpha at the cell membrane. However, only small fractions of cells were positive for intracellular IL-1 beta (less than 1%) and almost no IL-6-positive cells were found. We conclude that either IL-1 beta and IL-6 are produced by a minor population of undefined cells, or that a more sensitive in situ method is needed to detect production of these cytokines in B-CLL cells. The possible biological significance of secreted, and membrane-expressed helper factors in B-CLL is discussed.

Adult↗

PubMed1990

Stimulation of the T-cell receptors CD3 and CD2 with OKT3 and OKT11 antibodies activates a common pertussis toxin-insensitive G-protein.

The association of G-proteins with the T-cell-specific receptor structures CD3 and CD2 was investigated. High-affinity GTPase activity in membrane preparations of the human leukemic T-cell line Jurkat could be induced by the monoclonal antibodies OKT3 (anti-CD3) and OKT11 (anti-CD2). When combining maximally active concentrations of OKT3 and OKT11, no additive effect was seen on GTPase activity. In mutant Jurkat cells lacking the CD3 complex but with an intact CD2 receptor, neither OKT3 nor OKT11 could stimulate GTPase activity. Activation of CD3 and CD2 by monoclonal antibodies also stimulated phospholipase C activity as measured by breakdown of membrane phosphoinositides in wild-type but not in mutant Jurkat cells. Neither GTPase nor phospholipase C activation was sensitive to pretreatment with doses of pertussis toxin (PTX) that caused ADP ribosylation of a sensitive G-protein. Our data show that the CD3 complex and the CD2 receptor may activate a common PTX-insensitive G-protein. The CD2 receptor appears to stimulate the G-protein by interacting with the CD3 complex. The data are compatible with, but do not prove, that this G-protein is involved in the activation of phospholipase C by the two receptors.

Adenosine Diphosphate Ribose↗

PubMed1990

Agents that elevate cAMP stimulate DNA fragmentation in thymocytes.

Increases in the cAMP level are often inhibitory in mature T lymphocytes and may be involved in the development of tolerance to self Ag. In this report, agents inducing an increase in the cAMP level by independent mechanisms were found to stimulate DNA fragmentation, characteristic of a suicide program known as apoptosis, in isolated thymocytes. Data obtained with cAMP analogs known to act synergistically to stimulate protein kinase A suggested that the latter directly mediated endonuclease activation. Agents previously shown to stimulate protein kinase C and to inhibit Ca2(+)-dependent, TCR-mediated thymocyte apoptosis, including IL-1, also blocked both DNA fragmentation and cell death in response to cAMP, suggesting interactions ("cross-talk") between the two protein kinase systems. As it has been proposed that apoptosis mediates negative cell selection in the thymus, our results indicate that cAMP may play a role in the development of functional mature T lymphocytes.

Animals↗

PubMed1990

Regulation of calcium influx across the plasma membrane of the human T-leukemic cell line, JURKAT: dependence on a rise in cytosolic free calcium can be dissociated from formation of inositol phosphates.

A rise in the cytosolic free Ca2+ concentration due to both mobilization of Ca2+ from internal stores and influx of extracellular Ca2+ across the plasma membrane through 'second messenger-operated Ca2+ channels' is one of the first transmembrane signals detected following activation of CD2 or CD3 receptors on T-cells. In this study, we have further elucidated the regulation of these channels in the human T-leukemic cell line, JURKAT. Stimulation with either OKT3 or PHA induced a prompt influx of Ca2+ as assessed by MN2+ quenching of intracellular fura-2 fluorescence. When cytosolic free Ca2+ transient was partially buffered by loading the cells with BAPTA, neither agonist could induce Ca2+ entry into the cells as depicted by the lack of quenching of the fluorescence signal by Mn2+. This is in good agreement with our previous data on agonist-induced 45Ca2+ influx demonstrating that a rise in cytosolic free Ca2+ due to agonist-induced mobilization of Ca2+ from intracellular stores, could, directly or indirectly via the inositol cycle, initiate Ca2+ influx in these cells. Further support of this idea comes from the data demonstrating that agonist-induced mobilization of Ca2+ precedes the influx of Ca2+ across the plasma membrane. The present findings show that agonist-stimulation significantly increased the levels of Ins(1,4,5)P3 and Ins(1,3,4,5)P4 after only 5 s, indicating that one or both of these substances could play a role in the regulation of Ca2+ influx. However, when agonist-induced Mn2+ influx was totally abolished, by partially buffering the cytosolic free Ca2+ rise, the formation of Ins(1,4,5)P3 and Ins(1,3,4,5)P4 was not affected. Consequently, the dependence of an initial rise in cytosolic free Ca2+ for the subsequent regulation of Ca2+ influx across the plasma membrane, can be dissociated from the formation of both Ins(1,4,5)P3 and Ins(1,3,4,5)P4.

Aminoquinolines↗

PubMed1990

Inhibition of receptor-mediated calcium influx in T cells by unsaturated non-esterified fatty acids.

The effect of omega-3, omega-6 and omega-9 unsaturated fatty acids (UFAs) on receptor-mediated Ca2+ entry was investigated in a T-cell line (JURKAT) by using anti-CD3 antibodies (OKT3) to induce intracellular Ca2+ [( Ca2+]i) increase and Ca2+ influx. All the UFAs, as well as Ni2+ ions and 12-O-tetradecanoylphorbol 13-acetate, decreased the OKT3-induced sustained [Ca2+]i increase to basal levels. Although non-esterified fatty acids activate protein kinase C (PKC) [McPhail, Clayton & Snyderman (1984) Science 224, 622-624; Murakami, Chan & Routtenberg (1986) J. Biol. Chem. 261, 15424-15429], studies using H-7 and analysis of the PKC-dependent phosphorylation of 19 and 80 kDa marker substrates ruled out the involvement of PKC in UFA-induced inhibition of Ca2+ entry. Flow-cytometry analysis showed that UFAs do not interfere with antibody-receptor binding. BSA (0.2%, w/v) reversed the effect of UFAs after these fatty acids have decreased the OKT3-induced [Ca2+]i increase to basal levels. The relevance of these findings and possible mechanisms for inhibition by UFAs of receptor-mediated Ca2+ influx were discussed.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗