Klaus Resch

Ionophore A23187 raises cyclic AMP levels in macrophages by stimulating prostaglandin E formation.

Abstract The calcium ionophore A23187 rapidly increased cAMP in rat peritoneal macrophages. The cAMP accumulation required extracellular Ca 2+ and could be inhibited by indomethacin. Determinations of prostaglandins in the incubation medium revealed that the ionophore A23187 induced a rapid release of prostaglandin E (PGE) and prostaglandin F (PGF). Synthesis of PGE and PGF occurred only in the presence of extracellular Ca 2+ but was not found to depend on a translocation of Ca 2+ into the cells. The data suggest a direct perturbation of the cell membrane by the ionophore A23187, leading to a release of prostaglandins which mediate an increase of cAMP.

Membrane Associated Events in Lymphocyte Activation

Lymphocytes respond to a great number of substances (antigens) which can be recognized by surface receptors. Without being exposed to exter¬nal stimuli, lymphocytes of the blood or the peripheral lymphoid organs (lymph nodes, spleen) are small cells with only a basal metabolism, which do not proliferate or express apparent functions. When an antigen binds to the cells, a sequence of metabolic events is initiated resulting in growth and division, expansing the clone of cells, which reacts with the specific antigen. Concomitantly new functions are expressed. One subpopulation of lymphocytes starts to secrete specific antibodies (B, bursa-equivalent or bone marrow derived lymphocytes). A second subpopulation (T or thymus dependent lymphocytes) generates antigen sensitive cells which function as effectors in cell-mediated immune reactions or as regulators in antibody production [1–3]. In response to an antigen only a very restricted number of lymphocytes is activated. Thus it was a major advantage when it was first recognized by Nowell [4, 5] that in vitro phytohemagglutinin (PHA), a protein from Phaseolus vulgaris (the Red Kidney Bean), could also activate lymphocytes. In contrast to antigens, this mitogen induces mitosis in a high proportion of clones of resting lymphocytes.

Functional mosaicism of the lymphocyte plasma membrane. Characterization of membrane subfractions obtained by affinity chromatography on concanavalin A-Sepharose

Abstract Microsomal membranes (consisting largely of plasma membrane) or purified plasma membranes from calf thymocytes were fractionated by affinity chromatography on concanavalin A-Sepharose. Two fractions were obtained: one, designated MF1, eluted freely from the affinity adsorbent containing two thirds of the membrane protein. A second fraction, designated MF2, containing one third of the membrane protein, adhered to concanavalin A-Sepharose and was recovered after mechanical dissociation. Both fractions were homogeneous as assessed by rechromatography. The following control experiments suggested that the fractions were derived from different areas of an individual cell. (1) Fractionation required the binding of membranes to (Sepharose-bound) concanavalin A. (2) The non-retarded fraction MF1 exhibited more binding sites for concanavalin A, suggesting that both fractions were right side-out. (3) Intact thymocytes could not be fractionated on concanavalin A-Sepharose, which made it likely that the membrane subfractions did not originate from different cells. This was supported by an identical fractionation of plasma membranes isolated from a homogeneous T-lymphocyte tumor line. (4) Several membrane-bound enzymes (γ-glutamyltransferase, adenylate cyclase, Mg2+-ATPase) exhibited similar specific activities in both subfractions as well as in the unseparated membrane. In addition to previous findings that the cholesterol/phospholipid ratio was identical (Brunner, G., Ferber, E. and Resch, K. (1976) Differentiation 5, 161–164), this suggests that MF1 and MF2 consisted predominantly of identical membranes, i.e. the plasma membrane. The affinity for concanavalin A was 2.5 times higher in MF2 as compared to MF1. Both fractions were distinct in plasma membrane-bound enzymes: ( K + + Na + )-ATPase exhibited high specific activity in MF2 compared to MF1 or the unseparated membrane. Similarly, acyl-CoA:lysophosphatidylcholine acyltransferase was alos enriched in MF2. Alkaline p- nitrophenyl phosphatase showed also higher activity in this fraction. The data suggest a close association of receptors with high affinity for concanavalin A and certain membrane-bound enzymes. As activation of lymphocytes was initiated when 14–24% of the available binding sites interacted with concanavalin A, the data suggest a functional mosaicism in the vicinity of activating receptors.

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.

FUNCTIONAL MOSAICISM OF THE LYMPHOCYTE PLASMA MEMBRANE

Publisher Summary This chapter explores the functional mosaicism of the lymphocyte plasma membrane. Lymphocytes are activated to grow and divide when only a distinct proportion of plasma membrane-binding sites interacts with mitogens such as the lectin concanavalin A (ConA). To test whether binding sites responsible for cell activation are associated with specialized areas of the plasma membrane, calf thymocytes were disrupted by the nitrogen cavitation method and the microsomal membranes were isolated in a study described in the chapter. These vesicles, which are more than 90% derived from the plasma membrane, were subjected to affinity chromatography on ConA-sepharose. The results demonstrate that the plasma membrane of lymphocytes is not completely homogeneous. The areas of the plasma membrane that carry mitogen receptors, that is, high affinity binding sites, are distinct from the bulk membrane with regard to their enzymatic activities, suggesting a functional mosaicism in the vicinity of mitogen receptors.