Osawa Toshiaki

Effects of various mitogens on the phospholipid metabolism of human peripheral lymphocytes

Abstract The effects of various phytomitogens and anti-human lymphocyte horse serum on the phospholipid metabolism at the early stage of lymphocyte transformation were investigated. During the first 30 min treatment of human peripheral lymphocytes, all mitogens tested except pokeweed mitogen effected a selective acceleration (5-10-fold) in the rate of 32 PO 4 incorporation into phosphatidyl inositol, and an increased 32 PO 4 incorporation into other phospholipids was also observed after 3 h exposure to these mitogens. On the other hand, pokeweed mitogen did not affect 32 PO 4 incorporation into any phospholipids during the first 30 min of lymphocyte stimulation and, even in the prolonged treatment, more retarded and smaller 32 PO 4 incorporation into phospholipids was observed. These results suggest that the early acceleration of phosphatidyl inositol synthesis is one of the common biochemical events involved in the earliest stage of lymphocyte transformation at least by the mitogens which stimulate only thymus-derived cells.

Isolation and characterization of an inhibitory glycopeptide against guinea pig lymphotoxin from the surface of L cells

Abstract A glycopeptide which has inhibitory activity against guinea pig lymphotoxin (GLT) was isolated from the cell surfaces of L cells. It has an approximate mol. wt of 1600 (gel filtration) and contains galactose, mannose. fucose and N-acetylglucosamine in a molar ratio of 2:2:1:3. β-Galactosidase treatment of the glycopeptide significantly diminished its inhibitory activity against GLT. Furthermore. GLT was found to be inhibited by the lectins ( Ricinus communis haemagglutinin. Concanavalin A. and Phaseolus vulgaris haemagglutinin) which bind preferentially to sugar chains like those of serum glycoproteins (serum glycoprotein-type sugar chains) on the cell surface. These data indicate that serum glycoprotein-type sugar chains on the cell surface of target cells serve as receptors for GLT and that the β-galactosyl residue at the non-reducing terminal of the sugar chain is an important part of the receptor.

Poly[N-acetyl-lactosamine]-type sugar chains in CD45 antigens of abnormal T cells of 1pr mice are different from those of normal T cells and B cells

Abstract The lymph node (LN) T cells from autoimmune MRL/MpJ-lpr/lpr (lpr) mice and control MRL/MpJ-+/+ (+/+) mice were compared as to their cell surface lectin binding sites and the glycoproteins responsible for the lectin binding. T cells from enlarged lymph nodes of lpr mice were found to express more binding sites for lectins which are reactive to poly[ N -acetyl-lactosamine]-type sugar chains than normal +/+ mouse lymph node T cells. Furthermore, we found that high mol. wt (180,000–220,000) glycoproteins on lpr T cells were strongly stained with these poly[ N -acetyl-lactosamine]-binding lectins on Western-blotting. These glycoproteins were found to belong to the CD45 family on immunoprecipitation and absorption with monoclonal anti-CD45 antibody. Thus, aberrant expression of high mol. wt CD45 (CD45R) antigens on lpr T cells may contribute greatly to the strong reaction of these cells with poly[ N -acetyl-lactosamine]-binding lectins. We also found that poly[ N -acetyl-lactosamine]-type sugar chains are more abundant on B cells than on lpr T cells, and that the molecular weights and the carbohydrate moieties of CD45R antigens on Ipr T cells are different from those of CD45R antigens on +/+ spleen B cells.

Lectin-induced mitosis and phospholipid methylation☆

The binding of the mitogenic lectins concanavalin A, Wistaria floribunda mitogen, Pisum sativum hemagglutinin, Lens culinaris lectin, phytohemagglutinin, and two of the pokeweed mitogens (Pa-1 and Pa-2), in mitogenic concentrations to the cell surface acceptors of murine splenic lymphocytes caused a rapid, transient increase in the methylation of lymphocyte phospholipid. The extent of this increase was dependent upon the concentration of the lectin, and paralleled the dose-response curve for subsequent mitosis. Higher or lower doses of lectin did not cause the observed changes in methylation nor induce mitosis. The increase of phospholipid methylation reached a maximum about 10 min after the addition of the mitogenic lectins and then decreased to control levels within about 45 min. Accordingly, mitogenic lectins not only stimulate phospholipid methylation in lymphocytes, but also stimulate degradation of the phospholipids, probably by phospholipase A2. The non-mitogenic lectins, Sophora japonica hemagglutinins (Sj-I and Sj-II), W. floribunda hemagglutinin, and Bauhinea purpurea hemagglutinin did not significantly affect the phospholipid methylation. Preincubation of lymphocytes for 1 hr with 10−5M of the methylation inhibitor. S-isobutyryl-3-deazaadenosine, suppressed both the concanavalin A (Con A)-induced increase of phospholipid methylation after 10 min, and subsequent DNA synthesis after 45 hr. This concentration of methylation inhibidid not affect these responses if added immediately after the Con A stimulation, and appears to be specific for the phospholipid methylation. The degradation of methylated phospholipid was blocked by the phospholipase A2 inhibitor, mepacrine, which also suppressed DNA synthesis. The T-cell mitogen, Con A. showed the same lymphocyte specificity for phospholipid methylation enhancement as for stimulation of DNA synthesis. Con A caused the transient increase of phospholipid methylation in murine thymocytes, spleen cells treated with anti-immunoglobulin and complement, and purified T-cells. On the other hand, Con A did not cause a meaningful change of phospholipid methylation in spleen lymphocytes from athymic nude mice, spleen cells treated with rabbit anti-mouse brain and complement, or purified B-cells. From these results, it appears that both increased phospholipid methylation and degradation in mitogen-stimulated lymphocytes may be an integral part of the mitogenic signal.

Purification and cDNA cloning of a novel factor produced by a human T-cell hybridoma: sequence homology with animal lectins.

We have purified a novel immunoregulatory factor (BMPG: bone-marrow proteoglycan) produced by a T-cell hybridoma, with a monoclonal antibody column. Using an oligonucleotide probe corresponding to the partial amino acid sequence of BMPG, we cloned, sequenced, and expressed a cDNA for BMPG. BMPG has 222 amino acid residues with a 16 N-terminal signal sequence, so the mature form has 206 amino acid residues. BMPG was found to have unique characteristics: it has three types of sugar chains and it shows a marked homology with animal lectins including the human asialoglycoprotein receptor, chicken hepatic lectin and the homing receptor of lymphocytes.

Tnf-mediated cytotoxicity. importance of intracellular cgmp level for determining tnf-sensitivity☆

Previous work on the cytolytic action of activated macrophages indicated that tumor necrosis factor (TNF) showed synergistic cytolytic activity with NO, which has been shown to act as a cyclic GMP (cGMP) generator [Higuchi et al., J. Immun.144, 1425–1431, (1990)]. In this study, we investigated the relationship between the accumulation of intracellular cGMP and the cytotoxic action of TNF. It was demonstrated that TNF-mediated cell lysis was closely related to the background level of intracellular cGMP, and that the accumulation of cGMP within TNF resistant cells induced TNF sensitivity. We reached these conclusions on the basis of the following results; (1) agents (sodium nitroprusside and isobutylmethylxantine) that cause the accumulation of cGMP intracellularly increased the TNF-sensitivity of TNF-resistant cells; (2) the addition of dibutyryl cGMP to TNF-resistant cells increased the TNF-sensitivity; and (3) treatment at 40°C or agents such as interferon gamma and actinomycin D, that synergistically kill tumor cells together with TNF, potentially increased the cGMP level. Therefore, intracellular cGMP may be one of the key molecules that lead to cell death caused by TNF.

Enhancement of the activities of glycosyltransferases involved in the biosynthesis of mucin-type sugar chains in autoimmune MRL LPR/LPR mouse T cells

Abstract Lymph node (LN) T cells from autoimmune MRL/MpJ-Ipr/Ipr (lpr) mice and control MRL/MpJ-+ / + ( + / + ) mice were compared as to their cell surface lectin-binding sites and glycosyltransferase activities. T cells from enlarged LN of lpr mice expressed a higher amount of binding sites for lectins reactive to mucin-type sugar chains than normal +/+ mouse T cells. Correspondingly, glycosyltransferase activities involved in the biosynthesis of mucin-type sugar chains were higher in Ipr mouse T cells than in +/+ T cells. The activities of UDP-N-acetylgalactosamine (GalNAc):polypeptide GalNAc transferase and UDP-galactose (Gal):asialo bovine submaxillary mucin (BSM) Gal transferase were found to be elevated. The activity of UDP-Gal:asialo-agalacto transferrin Gal transferase, which is involved in the biosynthesis of complex type sugar chains, was also increased in Ipr mice but to a smaller extent than the mucin-type Gal transferase activities. An abnormality in sialyltransferase activity was also found in Ipr T cells.

A hapten-induced conformational change accompanies the cryoprecipitation of an immunoglobulin.

By simultaneous spectroscopic measurements (absorbance, light scattering, fluorescence and circular dichroism) the temp-dependent behavior of a cryoglobulin, the mouse monoclonal immunoglobulin G to N-acetyllactosamine, has been observed. The results show that a hapten-induced conformational change in the structure of a cryoglobulin can accompany its intermolecular association. This association of the immunoglobulin increases after hapten saturation of its two antigen-combining sites.

Establishment of a human T-Cell hybridoma that produces human macrophage activating factor for superoxide production and translation of messenger RNA of the factor in Xenopus Laevis oocyte

Abstract Human monoblast-like histiocytic lymphoma cell line U937 was induced by a macrophage activating factor for O 2 − production (MAF-O) to produce O 2 − in response to phorbol myristate acetate stimulation. A MAF-O-producing human T-cell hybridoma, F4-29-4, was established which was also found to produce macrophage activating factor for glucose consumption (MAF-G) and colony stimulating factor (CSF) when assayed against mouse bone marrow cells. MAF-O could be successfully separated from CSF but not from MAF-G by phenyl-Sepharose CL-4B chromatography (Phenyl-EG-fraction). To differentiate MAF-O from MAF-G and to explore a route for large scale production of MAF-O and its structural elucidation, total messenger RNA was extracted from a human T-cell hybridoma, clone F4-29-4. This messenger RNA was fractionated on 5–30% sucrose gradient and each fraction was microinjected into Xenopus laevis oocytes. MAF-O activity was found in the supernatant of oocytes injected with messenger RNA sedimentated at about 13.0S, while MAF-G messenger RNA was found to be about 10.5S. The MAF-O activity, synthesized from the injected messenger RNA, was not neutralized with an excess amount of anti-human IFN-γ anti-serum, suggesting that MAF-O is antigenically different from human IFN-γ.

Purification and characterization of a mouse t cell replacing factor from primary mixed lymphocyte culture supernatant.

Abstract The purification and chemical characterization of a T cell replacing factor (TRF), which was induced in the supernatant of primary mixed lymphocyte cultures for 24 hr under serum-free conditions, has been carried out. This TRF was sensitive to proteolytic enzymes, trinitrobenzene sulfonic acid and sodium metaperiodate, while it was stable in buffers of a wide pH range (pH 5–10) and to freezing and thawing treatment. After purification of the TRF by DEAE Sephadex A-50 ion-exchange chromatography, preparative disc electrophoresis and gel filtration on Sephadex G-100, the final product revealed only one band after sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Chemical analyses of the purified TRF showed that it was a protein, abundant in serine, valine, glycine, glutamic acid, almost devoid of basic amino acids and that it contained approximately 10% carbohydrate. Furthermore, the purified TRF described in this paper was found to consist of two identical subunits of molecular weight of 21,000 ± 3000 daltons.