H. Northoff

Ligand-dependent modulation of membrane phospholipid metabolism in ConA-stimulated lymphocytes

Abstract Human peripheral lymphocytes were activated by ConA in serum-free culture medium, supplemented by BSA. Incorporation of [3H]thymidine into DNA, of [3H]uridine into RNA and of oleate or acetate into membrane phospholipids was investigated. DNA synthesis could be completely inhibited by αMM or by anti-ConA-IgG. Fab and F(ab)2 fragments of the anti-ConA were equally active. When αMM or anti-ConA was added to cultures at different times after stimulation with ConA, incorporation of [3H]thymidine into DNA (measured after 72 h) could be prevented up to 6–8 h completely and up to 20–30 h partially. Incorporation of [3H]uridine into RNA could be arrested at any time of the culture up to 40 h at the level it had reached but did not reverse to the level of unstimulated cells for a long time. In contrast, incorporation of oleate into lecithin returned to the level of unstimulated cells within 2–3 h after removal of ConA. This suggests that the activation of the phospholipid turnover in stimulated cells is a direct consequence of the presence of the mitogen at the membrane and thus may be a critical initial event in lymphocyte activation.

Antibody Dependent Cellular Cytotoxicity (ADCC) against Human Erythrocytes, Mediated by Blood Group Alloantibodies: A Model for the Role of Antigen Density in Target Cell Lysis

Antibody dependent cellular cytotoxicity (ADCC) of human mononuclear cells against human erythrocytes could be obtained with anti-A and anti-D sera. The degree of lysis varied considerably depending on the antigen system and on the experimental conditions. Anti-D mediated in contrast to anti-A mediated ADCC turned out to be very sensitive to conditions which interfere with target cell lysis: for most of the anti-D sera, removal of unbound IgG was found to be crucial to detect their ability to mediate ADCC. Pretreatment of the target cells with various enzymes dramatically improved specific lysis and left spontaneous release and spontaneous cytotoxicity essentially unaffected. In the case of neuraminidase treatment it could be shown that the effect was independent from the exposure of additional binding sites. When enzyme treatment and removal of excess IgG were applied in combination, as little as 10(4) antigenic determinants proved to be sufficient to induce specific lysis.

The effect of anti-ConA on the binding of ConA to lymphocytes.

Abstract Fab and F(ab) 2 fragments were prepared from the IgG fraction of a rabbit antiserum to concanavalin A (ConA). Both fragment preparations, which were tested for purity by immunodiffusion and agglutination techniques, showed identical capacity to suppress the incorporation of [ 3 H]uridine and [ 3 H]thymidine into ConA stimulated lymphocytes as compared with intact IgG. Binding of [ 125 I]ConA to lymphocytes was measured using centrifugation of the cells through an olive oil/phthalate gradient. Intact IgG at concentrations suboptimal for the suppression of lymphocyte activation decreased the binding of [ 125 I]ConA. At optimal or higher concentrations, cell-associated ConA was strongly increased. In contrast, the Fab and the F(ab) 2 fragments both prevented or reversed binding over the whole dose range. We conclude that all three preparations prevented binding of ConA to its receptors, that, however, intact IgG in the equivalence dose or moderate antibody excess lead to the formation of ConA-anti-ConA complexes which adhere to the lymphocytes via the Fc receptor.

Induction of the proteinase inhibitor alpha 2-macroglobulin in rat hepatocytes by a monocyte-derived factor.

Disturbances of the physiologic homeostasis such as infections, tissue injury, tumor growth and immunologic disorders lead to a highly complex reaction of the organism, the so called acute-phase response1-3. The acute-phase response is characterized by fever, leukocytosis, a negative nitrogen balance, depression of serum iron and zinc levels, elevation of serum copper and dramatic changes in the synthesis of hepatic acute-phase proteins4,5. The plasma concentration of the proteinase inhibator α2-macroglobulin (α2M) or example increases 100-500-fold in the rat during acute inflammation. The concentration of the proteinase-inhibitor α1-inhibitor3 (α1I3) belonging to the same macroglobulin family decreases to about 30% simultaneousely. These changes are generated by mediators secreted from mononuclear phagocytes. Hepatocyte-stimulating factor (HSF), interleukin 1 (IL-1), tumor necrosis factor α (TNFα) and interferon s (IFN s) are the most important of theses mediators.