M. Sándor

Immune-complex-induced transglut aminase activation: Its role in the Fc-receptor-mediated transmembrane effect on peritoneal macrophages

Abstract The binding of soluble immune complexes (IC) or haemolysin-sensitized erythocytes (EA) to the Fc receptor of rat peritoneal macrophages was followed by a rapid increase of macrophage transglutaminase activity measured in cell homogenates and a time-dependent incorporation of 14 C-methylamine into proteins of the intact cells. Methylamine, a competitive substrate inhibitor of transglutaminase could inhibit EA rosette formation as well as the soluble IC- or EA-induced lipid reordering of the plasma membrane of macrophages. Cytochalasin B (CB) which prevents EA rosette formation as well as IC-induced lipid reordering did not affect the stimulation of transglutaminase activity by IC. The possible relation of transglutaminase activation, lipid reordering and the contractile system to each other in the IC (multivalent ligand)-induced Fc receptor redistribution is discussed.

The influence of tissue transglutaminase on the function of Fc receptors

In contrast to FcRII the soluble Fc receptor (FcRI) of human peripheral mononuclear blood cells (PMBC) is shed from PMBC following a 4-37 degrees C temperature shift and inhibits rosette formation of nonshed PMBC with antibody-coated erythrocytes (EA). Purified FcR, could be polymerized by tissue transglutaminase as was revealed by SDS-polyacrylamide gel electrophoresis. Comparing the Sephadex G-150 elution profile of the EA rosette inhibitory capacity of FcRI vs FcRI incubated in the presence of transglutaminase, the latter was found in a higher mol. wt region and could inhibit rosette formation by both FcRI and FcRII. Furthermore, the shedding of FcRI could be prevented by the addition of transglutaminase or Ca2+-ionophore A23187 (which leads to the activation of PMBC transglutaminase) to the cell suspension. The function of FcRII was not affected by either the addition of transglutaminase or Ca2+-ionophore to the cells. The results point to the involvement of transglutaminase in the determination of the functional state of the Fc receptor on the cell surface.

Interaction between actomyosin complexes and Fc receptors on human peripheral mononuclear blood cells

Abstract The soluble FcRI shed from HPMBC following 4–37°C temperature shift interacts with AM, A-HMM and A-S1, respectively, but do not bind to A, M, HMM or S1. In contrast to the monovalent soluble FcRI the multivalent A-HMM-FcRI and A-S1-FcRI complexes agglutinate EA cells. Due to the interaction of soluble FcR with muscle proteins an alteration in the binding properties of the receptor was observed. FcRI in soluble form inhibits only the EA rosette formation of FcRI + HPMBC in contrast to FcRI-A-HMM and FcRI-A-S1 complexes inhibiting the rosette formation of FcRI + and FcRII + cells as well. Based on these observations one can suppose that depending on their anchoring to cytoskeletal structures the FcRs possess one or two binding sites.

Immune complex binding induces lateral lipid re-ordering in rat macrophage plasma membrane.

Abstract The plasma membrane of rat peritoneal exudate cells was spin labelled with fatty acid spin probes and structural changes induced by ligand binding to Fc and C3b receptors were followed. Neither antigens, nor monovalent immune complexes were capable of inducing any change. Multivalent ligands, both in soluble and particulate form, and to a lesser extent uncomplexed immunoglobulins were very effective in inducing membrane re-ordering, i.e. a decrease in fluidity. The phase transition region markedly broadened upon binding of multivalent immune complexes, indicating phase separation. There was a time lag of 5–10 min before the onset of lipid re-ordering was observed. We suggest that multivalent binding that is capable of cross-linking surface receptors initiates lateral phase separation and lipid re-ordering due to receptor aggregation.

Changes in the lateral ordering of the macrophage plasma membrane during Fc receptor mediated phagocytosis.

The macrophage plasma membrane was labelled with an intercalated 5-doxyl stearic acid spin probe, and structural changes induced by IgG-coated erythrocytes (EA) were followed with particular emphasis on the possible role of lipid reordering in the sequential events of phagocytosis. We present three lines of experimental evidence to show that these structural changes were induced by the lateral aggregation of cell surface Fc receptors. Cytochalasin B, an inhibitor of microfilament function, blocked this membrane reordering; if it was added after EA binding induced membrane reordering had already been detected for 15 min, a rapid reversal process was observed resulting in a reversible restoration of the initial order parameter value. We suggest that these structural changes indicate lipid-lipid lateral phase separation, in line with morphological findings.

Receptors for IgM on rat spleen cells.

Abstract Ox red blood cells (ORBCs) sensitized with rat IgM antibodies formed antibody-coated red blood cell rosettes (EAR) around 9% of rat spleen cells freshly prepared at 4 °C, while an enhancement of the rosette-forming cell (RFC) frequency to 18–20% was observed after having exposed the spleen cells to a temperature shift from 4 to 37 °C. Although the temperature shift was found to increase the RFC frequency, a shedding of IgM-Fc receptors IgM-FcR into the medium was also detected (IgM-FcR-I). Regeneration of shed receptors within 5 hr has been proved, and the sheading-regeneration cycle could be repeated several times. IgM-Fc receptors detectable after the temperature shift (IgM-FcR-II) are neither shed nor detectable on freshly prepared spleen cells. This latter type of receptor is expressed only after incubating the cells at 37 °C for an optimal period of 8–10 hr. Both type I and II IgM-FcRs were detected on T lymphocytes as well as B lymphocytes; therefore they do not mark subpopulation. Both receptors are sensitive to trypsin and the activity of both requires Ca2+ ions. Shed receptors are able to inhibit EAR formation by cells carrying either IgM-FcR-I or IgM-FcR-II. They are sensitive to higher temperatures and agglutinate ORBCs sensitized by IgM antibodies in the presence of Ca2+ ions. EAR-inhibiting capacity was detected in two fractions obtained by gel chromatography of the supernatant after shedding. The elution volume of one of the active fractions corresponds to 130,000 daltons (D), and that of the other to approximately 50,000 D.

The binding of IgG1 containing immune complexes to the FcR of allogenically activated T cells induces changes in the membrane potential and the cell surface charge

The effect on membrane potential and cell surface charge of binding immune complexes containing IgG1 and IgG2a monoclonal antibodies to Fc receptors was studied in resting and allogenically activated murine T cells. IgG1 complexed by antigen or heat aggregation induced electrophysiological changes on activated T cells. A biphasic alteration of membrane potential was detected by measurement of the intra- and extracellular distribution of the fluorescent dye, DiOC6. A short-lived hyperpolarization, detectable for 4-6 min after adding the respective ligand, was followed by a longer lasting depolarization. The cell surface charge, measured by cell electrophoresis, was also changed. This alteration was detected 2-4 hr after addition of immune complexes and disappeared by the 8th hr of incubation. Monoclonal antibody 2.4.G2, reactive with mouse FcR, induced a similar membrane potential response on activated T cells, but did not affect the cell surface charge. Monomeric IgGs and complexes of IgG2a did not modify these parameters. FcR ligands had no effect on the studied characteristics of resting T cells.

Effect of binding of C3 and its fragments on the plasma membrane fluidity of lymphocytes

Abstract In our previous papers the complement-dependent inhibition of Fc receptors (FcRs) on human peripheral blood mononuclear cells (PMBCs) was described. The inhibition of FcRs was shown to be induced by C3b fragments fixed to the acceptor sites on the cells. To explain the mechanism of this phenomenon different models have been proposed. In the present investigations the effect of C3b, nascent C3b and aggregated C3 on the membrane fluidity of PMBCs was studied using intercalated spin probes. C3b molecules did not affect membrane fluidity when bound to C3b acceptor sites or to C3b receptors. On the other hand, binding of aggregated C3 decreases the membrane fluidity without inhibiting EA rosette formation. It can be concluded that fixing of nascent C3b fragments does not inhibit FcRs via fluidity modulation.

IgG-Fc receptors differ in sensitivity to primary amines

The effect of 12 different amines was tested on the IgG-Fc receptor (IgG-FcR) function of human peripheral mononuclear blood cells (PMBCs). While histamine and to a lesser extent dopamine resulted in a higher ratio of EA-rosette forming cells, a group of amines, like methylamine, dansylcadaverine, hydroxylamine and some others inhibited the EA-rosette formation. Neither the enhancement nor the inhibition was found to be mediated through direct interaction of amines with the Fc portion of the IgG molecule or the IgG-FcR. Only a portion of IgG-FcRs are sensitive to the amine-mediated inhibition and the results suggest that amines inhibit the cluster formation of IgG-FcRs.