Fumihiko Nagase

Expression of chimeric receptor composed of immunoglobulin-derived V regions and T-cell receptor-derived C regions

Chimeric genes composed of immunoglobulin (Ig)-derived variable (V) regions and T-cell receptor (TCR)-derived constant (C) regions were constructed. The VL and VH genes showing anti-phosphorylcholine (PC) activity were used in this study. Two pairs of chimeric genes, VL-C beta and VH-C alpha genes, and VL-C alpha and VH-C beta genes, were inserted into an expression vector containing both Ecogpt and neo genes, and transfected into EL4 cells. Cells which express both chimeric receptor molecules were established. The activity of the transformants to the antigen was examined by using stopped-flow fluorometry. An increase in the concentration of cytoplasmic calcium ion was observed after addition of Staphylococcus pneumoniae R36A bacteria grown in the choline-containing medium which express PC molecules, but not after the PC-negative bacteria grown in the ethanolamine-containing medium.

Superoxide-mediated early oxidation and activation of ASK1 are important for initiating methylglyoxal-induced apoptosis process

Methylglyoxal (MG) is a physiological metabolite, but it is known to be toxic, inducing stress and causing apoptosis. Our previous studies demonstrated that MG induced apoptosis in Jurkat cells by activating the c-Jun N-terminal kinase (JNK) signal transduction pathway, which induced an obvious decrease in mitochondrial membrane potential, followed by caspase-3 activation. Here, we observed that MG-induced apoptosis was associated with both rapid production of superoxide anion (O(2)(-)) followed by a marked increase in ROS and striking and temporal activation of ASK1. Overexpression of wild-type ASK1 could enhance the rate of apoptosis induced by MG, whereas the expression of the kinase-inactive form of ASK1 notably prevented cells from MG-induced death. NAC and PDTC blocked the activation of ASK1 and MG-induced apoptosis completely. Moreover, nonthiol antioxidants SOD-mimic MnTBAP and catalase together obviously inhibited MG-induced ASK1 activation and apoptosis induction. Correspondingly, MG-mediated ASK1 activation was enhanced by diethyldithiocarbamate (DDC). Addition of antioxidant into the culture of cells at a later stage (4-8 h after the initial MG treatment) failed to prevent their death. These results suggest that activating ASK1 at the early stage linking to production of O(2)(-) is crucial for subsequent progression of apoptosis in MG-treated Jurkat cells.

Methylglyoxal induces apoptosis in Jurkat leukemia T cells by activating c‐Jun N‐Terminal kinase

Methylglyoxal (MG) is a physiological metabolite, but it is known to be toxic, inducing stress in cells and causing apoptosis. This study examines molecular mechanisms in the MG‐induced signal transduction leading to apoptosis, focusing particularly on the role of JNK activation. We first confirmed that MG caused apoptosis in Jurkat cells and that it was cell type dependent because it failed to induce apoptosis in MOLT‐4, HeLa, or COS‐7 cells. A caspase inhibitor, Z‐DEVD‐fmk, completely blocked MG‐induced poly(ADP‐ribose)polymerase (PARP) cleavage and apoptosis, showing the critical role of caspase activation. Inhibition of JNK activity by a JNK inhibitor, curcumin, remarkably reduced MG‐induced caspase‐3 activation, PARP cleavage, and apoptosis. Stable expression of the dominant negative mutant of JNK also protected cells against apoptosis notably, although not completely. Correspondingly, loss of the mitochondrial membrane potential induced by MG was decreased by the dominant negative JNK. These results confirmed a crucial role of JNK working upstream of caspases, as well as an involvement of JNK in affecting the mitochondrial membrane potential. J. Cell. Biochem. 77:333–344, 2000.

Further Studies of the Polysaccharide of Klebsiella pneumoniae Possessing Strong Adjuvanticity: I. Production of the Adjuvant Polysaccharide by Noncapsulated Mutant

In culture fluid, Klebsiella pneumoniae type 1 Kasuya strain produces polysaccharide exhibiting a strong adjuvant effect. The active substance responsible for the strong adjuvant effect of the polysaccharide is not its acidic polysaccharide fraction (the type‐specific capsular antigen) but the neutral polysaccharide fraction. In the present study, a mutant which did not produce the type‐specific capsular polysaccharide was isolated from ultraviolet‐irradiated cells of K. pneumoniae type 1 Kasuya strain which had been labeled with leucine‐requiring marker by selecting unagglutinable cells with the antiserum to the type‐specific capsular polysaccharide. Serological tests showed that the type‐specific acidic capsular polysaccharide was present neither on the cells surface nor in the culture fluid of the mutant. Electron microscopically, the mutant did not possess any capsular material. On the other hand, nearly an equal amount of neutral polysaccharide antigen was produced in culture fluids of the noncapsulated mutant and the parent strain. The neutral polysaccharide antigen produced by the noncapsulated mutant exhibited the same degree of strong adjuvant effect on antibody response to bovine gammaglobulin in mice as that produced by the parent strain. The relationship between the neutral polysaccharide antigen in culture fluid and the O antigen of K. pneumoniae was discussed.

Adjuvant actions of polyclonal lymphocyte activators: I. Comparison and characterization of their actions in antibody response to deaggregated bovine serum albumin

Abstract Various polyclonal lymphocyte activators (PLA) were compared in their effects to trigger the initiation of the immune response and the amplification of the once-initiated immune response. When PLA were injected into mice subcutaneously together with deaggregated bovine serum albumin (DBSA), all of the nine kinds of PLA tested, such as capsular polysaccharide of Klebsiella pneumoniae (CPS-K), lipopolysaccharide, dextran sulfate, concanavalin A, pokeweed mitogen, phytohemagglutinin, polyadenylic-uridylic acid, and polyinosinic-cytidylic acid, exhibited more or less the adjuvant action to trigger the antibody response and to increase immunological memory to DBSA. Among the PLA tested the action of CPS-K was the strongest. On the other hand, none of these PLA, including CPS-K, acted to augment the secondary antibody response to the optimal dose of DBSA in mice primed with DBSA together with CPS-K. It was concluded that PLA act generally to trigger the initiation of the antibody response, although the intensity of their actions distributes in a wide range of diversity, but that they do not stimulate the amplification of the response.

Hemoglobin induces the expression of indoleamine 2,3‐dioxygenase in dendritic cells through the activation of PI3K, PKC, and NF‐κB and the generation of reactive oxygen species

Indoleamine 2,3‐dioxygenase (IDO) is the rate‐limiting enzyme in the kynurenine (Kyn) pathway of tryptophan (Trp) metabolism. IDO is immunosuppressive and is induced by inflammation in macrophages and dendritic cells (DCs). Previous studies have shown the serum Kyn/Trp levels in patients with hemolytic anemia to be notably high. In the present study, we demonstrated that hemoglobin (Hb), but not hemin or heme‐free globin (Apo Hb), induced IDO expression in bone marrow‐derived myeloid DCs (BMDCs). Hb induced the phosphorylation and degradation of IκBα. Hb‐induced IDO expression was inhibited by inhibitors of PI3‐kinase (PI3K), PKC and nuclear factor (NF)‐κB. Hb translocated both RelA and p52 from the cytosol to the nucleus and induced the intracellular generation of reactive oxygen species (ROS). Hb‐induced IDO expression was inhibited by anti‐oxidant N‐acetyl‐L‐cysteine (NAC) or mixtures of SOD and catalase, however, IDO expression was enhanced by 3‐amino‐1,2,4‐triazole, an inhibitor of catalase, suggesting that the generation of ROS such as O  2− , H2O2, and hydroxyl radical is required for the induction of IDO expression. The generation of ROS was inhibited by a PKC inhibitor, and this action was further enhanced by addition of a PI3K inhibitor. Hb induced Akt phosphorylation, which was inhibited by a PI3K inhibitor and enhanced by a PKC inhibitor. These results suggest that the activation of NF‐κB through the PI3K‐PKC‐ROS and PI3K‐Akt pathways is required for the Hb‐induced IDO expression in BMDCs. J. Cell. Biochem. 108: 716–725, 2009.

Adjuvant actions of polyclonal lymphocyte activators: II. Comparison and characterization of their actions in initiation and potentiation of immune responses to T-dependent and T-independent soluble antigens

Abstract Various polyclonal lymphocyte activators (PLA) such as capsular polysaccharide of Klebsiella pneumoniae (CPS-K), lipopolysaccharide of Escherichia coli (LPS), dextran sulfate (DS), concanavalin A (Con A), phytohemagglutinin (PHA), pokeweed mitogen (PWM), and polyadenylic-polycytidylic acid (poly A:U) were compared in their effects on antibody response to T-dependent antigen (bovine gamma globulin (BGG) and dinitrophenylated (DNP)-BGG) and T-independent antigen (DNP-Ficoll) and on induction of tolerance to T-dependent antigen. All of these PLA acted more or less to trigger the initiation of the antibody-forming mechanism for deaggregated BGG (DBGG) or DNP-BGG through their actions on the carrier-specific T-cell function. All of these PLA tested also acted more or less to inhibit the induction of the carrier-specific T-cell tolerance to DBGG. Moreover, some of these PLA could act to augment antibody response to DNP-Ficoll. The adjuvant action of PLA in the response to DNP-Ficoll worked as well in athymic nu/nu mice as in nu /+ mice, whereas that in the response to DNP-BGG did not occur in athymic nu/nu mice. The order of the strength of the action of PLA to trigger the initiation of the whole immune response to DBGG, that to trigger the carrier-specific T-cell function to DNP-BGG, and that to inhibit the induction of the whole tolerance to DBGG was very similar to each other: i.e., CPS-K ⪢ Con A > LPS, DS, poly A:U, PWM and PHA. By contrast, the order of the strength of the action to inhibit the induction of T-cell tolerance to DBGG was ≧ = LPS > Con A, PWM and poly A:U > DS and PHA, and that of the action to augment the antibody response to DNP-Ficoll was CPS-K > LPS > Con A. CPS-K was the most potent in all of these immunological activities. It was concluded that PLA act generally to stimulate the immune response at its initiation step in which T cells in the case of T-dependent antigen and B cells in the case of T-independent antigen play a predominant role, but that individual PLA share this adjuvant activity in different fashions.

Involvement of MEKK1/ERK/P21Waf1/Cip1 signal transduction pathway in inhibition of IGF-I-mediated cell growth response by methylglyoxal.

The abnormal accumulation of methylglyoxal (MG), a physiological glucose metabolite, is strongly related to the development of diabetic complications by affecting the metabolism and functions of organs and tissues. These disturbances could modify the cell response to hormones and growth factors, including insulin‐like growth factor‐1 (IGF‐I). In this study, we investigated the effect of MG on IGF‐I‐induced cell proliferation and the mechanism of the effect in two cell lines, a human embryonic kidney cell line (HEK293), and a mouse fibroblast cell line (NIH3T3). MG rendered these cells resistant to the mitogenic action of IGF‐I, and this was associated with stronger and prolonged activation of ERK and over‐expression of P21Waf1/Cip1. The synergistic effect of MG with IGF‐I in activation of ERK was completely abolished by PD98059 but not by a specific PI3K inhibitor, LY294002, or a specific PKC inhibitor, bisindolylmaleimide. Blocking of Raf‐1 activity by expression of a dominant negative form of Raf‐1 did not reduce the enhancing effect of MG on IGF‐I‐induced activation of ERK. However, transfection of a catalytically inactive form of MEKK1 resulted in inactivation of the MG‐induced activation of ERK and partial inhibition of the enhanced activation of ERK and over‐expression of p21Waf1/Cip1 induced by co‐stimulation of MG and IGF‐I. These results suggested that the alteration of intracellular milieu induced by MG through a MEKK1‐mediated and PI3K/PKC/Raf‐1‐independent pathway resulted in the modification of cell response to IGF‐I for p21Waf1/Cip1‐mediated growth arrest, which may be one of the crucial mechanisms for MG to promote the development of chronic clinical complications in diabetes. J. Cell. Biochem. 88: 1235–1246, 2003.

Cinnabarinic acid generated from 3‐hydroxyanthranilic acid strongly induces apoptosis in thymocytes through the generation of reactive oxygen species and the induction of caspase

3‐Hydroxyanthranilic acid (3HAA) is one of the tryptophan metabolites along the kynurenine pathway and induces apoptosis in T cells. We investigated the mechanism of 3HAA‐induced apoptosis in mouse thymocytes. The optimal concentration of 3HAA for apoptosis induction was 300–500 µM. The induction of apoptosis by a suboptimal concentration (100 µM) of 3HAA was enhanced by superoxide dismutase (SOD) as well as MnCl2 and further promoted in the presence of catalase. The 3HAA‐mediated generation of intracellular reactive oxygen species (ROS) was enhanced by SOD or MnCl2 and inhibited by catalase. Corresponding to apoptosis induction, the generation of cinnabarinic acid (CA) through the oxidation of 3HAA was enhanced by SOD or MnCl2 in the presence of catalase. The synthesized CA possessed more than 10 times higher apoptosis‐inducing activity than 3HAA. The intracellular ROS generation was induced by CA within 15 min and decreased to the control levels within 4 h, whereas the 3HAA‐induced ROS generation increased gradually up to 4 h. Corresponding to ROS generation, the mitochondrial membrane potential was downregulated within 15 min and retained by the CA treatment. Apoptosis induction by 3HAA or CA was dependent on caspases, and caspase‐3 was much more strongly activated by CA than 3HAA. In conclusion, the CA generated from 3HAA possesses a strong apoptosis‐inducing activity in thymocytes through ROS generation, the loss of mitochondrial membrane potential, and caspase activation. J. Cell. Biochem. 103: 42–53, 2008.

Redox-Linked Cell Surface-Oriented Signaling for T-Cell Death

T-cell death, which occurs either for ontogenic T-cell selection or for activated T-cell elimination, is normally induced through binding of a specific ligand to cell-surface T-cell receptor for crosslinkage. Heavy metals and carbonyl compounds that bind to protein-reactive groups such as cysteine sulfhydryl groups and lysine epsilon-amino groups may also induce crosslinkage of cell-surface proteins, in part replacing or modifying the ligand-mediated action. This chemical event has been found to accompany clustering of membrane rafts, to which signal-transducing elements such as glycosylphosphatidylinositol-anchored proteins and Src family protein tyrosine kinases (PTKs) are attached, and to trigger the signal transduction for apoptotic T-cell death, inducing mitochondrial membrane potential reduction, caspase activation and DNA fragmentation. As signals potentially upstream of this signaling, activations of PTKs and mitogen-activated protein (MAP) family kinases and production of reactive oxygen species (ROS) were induced following the cell-surface event, and crucial roles of activation of c-Jun amino-terminal kinase and apoptosis signal-regulating kinase 1 by a redox-linked mechanism in the cell-death signaling were demonstrated. Intriguingly, ROS production as well as PTK/MAP family kinase activation occurred in a membrane raft integrity-dependent manner. The redox-linked and cell surface-oriented signal delivery pathway demonstrated here may play an important role in induction of immune disorders by protein reactive group-binding chemicals.