Zoltan Rozsnyay

Mapping and comparison of the interaction sites on the Fc region of IgG responsible for triggering antibody dependent cellular cytotoxicity (ADCC) through different types of human Fcγ receptor

In the present study 3-iodo-4-hydroxy-5-nitrophenacetyl (NIP)-specific antibodies were compared for induction of antibody dependent lysis of NIP-derivatised red blood cells effected by pre-stimulated U937 or HL-60 cells and by K cells. The chimaeric antibodies have heavy chains corresponding to human IgG subclasses 1-4, and include site-directed mutants of IgG3 as well as the aglycosylated form of IgG3; a mouse IgG2b antibody and a site-directed mutant IgG2b were also examined. rIFN stimulated U937 or HL-60 cells express increased levels of Fc gamma R1 compared to unstimulated cells; PMA stimulated HL-60 and U937 cells express an increased level of Fc gamma R11 compared to unstimulated cells; K cells express Fc gamma R111. Using these effector cell populations and the target cells mentioned above, we have compared anti-NIP antibodies with different heavy chain constant domains for their ability to induce ADCC through human Fc gamma R1, Fc gamma R11 and Fc gamma R111. The results suggest that all three human Fc gamma receptors appear to recognise a binding site on IgG within the lower hinge (residues 234-237) and trigger ADCC via this site, but that each receptor sees this common site in a different way. The possibility that other amino acid residues also participate in the binding/triggering site(s) cannot be excluded.

Phenylarsine oxide (PAO) blocks antigen receptor-induced calcium response and tyrosine phosphorylation of a distinct group of proteins

Antigen receptor (AgR) crosslinking by antigens or AgR-specific antibodies induces a cascade of enzymatic events in lymphocytes which involves activation of several non-receptor tyrosine- and serine/threonine kinases, phosphatases, phospholipases, etc. Here we show data demonstrating that a thiol group-reactive protein tyrosine phosphatase (PTP) inhibitor, phenylarsine oxide (PAO), uncouples a crucial part of the signaling events induced by anti-IgM or anti-Leu-4 (CD3) in human tonsil B lymphocytes, BL41 and Daudi B cell lines and Jurkat T lymphoma cells. PAO treatment (10 microM) resulting in distinct modification of AgR-induced tyrosine phosphorylation pattern inhibited the AgR-mediated calcium response (Ca++ release and influx) of all of these cells completely. Since this treatment did not alter the cell viability and the binding capacity of the AgR crosslinking antibodies, alteration of the tyrosine phosphorylation pattern and blockage of the calcium response indicate prompt inactivation of essential signal transduction element(s).

Interaction of signaling molecules with human FcγRIIb1 and the role of various FcγRIIb isoforms in B-cell regulation

Abstract The low-affinity type-IIb IgG Fc-binding receptors (FcγRIIb) are expressed on B cells. When cross-linked with mIgM FcγRIIb are known to down-regulate B-cell activation by interrupting signal transduction upstream from G-protein-activated events. We have studied FcγRII isoforms expressed on resting and activated B cells and the interaction of FcγRIIb1 with molecules transducing the antigen receptor-mediated signals. Expression of FcγRII isoforms was studied by reverse transcription and polymerase chain reaction. Resting B cells express both FcγRIIb2 and FcγRIIb1 isoforms. Activation with anti-IgM or IL-4 induces the splicing of FcγRIIb1 mRNA, while the alternative splicing of FcγRIIb2 mRNA is down-regulated, resulting in the surface expression of FcγRIIb1. Functional differences were found between the two isoforms in inhibiting B-cell activation, suggesting that FcγRIIb2 might influence the threshold of signals necessary for activation of resting B cells, while FcγRIIb1 may regulate in later phases of antibody response. To explore the mechanism by which FcγRII may uncouple antigen receptor-mediated signal transduction, we have investigated the association of signaling molecules with FcγRII. Beside the protein tyrosine kinase (PTK) fyn, protein kinase C (PKC) was found to be co-isolated with FcγRIIb1, suggesting a tight connection between these kinases and FcγRII. We suggest that PKC might be responsible for the activation-induced phosphorylation of FcγRII on serine residues. Signaling molecules responsible for the activation and localization of further elements of the activation pathway were also found to associate with FcγRII. Among these RasGAP and Shc, the adapter molecule connecting PTK-triggered events to the Ras activation-dependent pathway were characterized. We suggest that FcγRII may compete with the B-cell antigen receptor for key molecules regulating Ras activity, inhibiting thereby Ras activation.

Rapid desensitization of B-cell receptor by a dithiol-reactive protein tyrosine phosphatase inhibitor: uncoupling of membrane IgM from syk inhibits signals leading to Ca2+ mobilization

B-cell antigen receptor (BCR)-mediated calcium response can be blocked by phenylarsine oxide (PAO), a dithiol group-reactive protein tyrosine phosphatase inhibitor. We have examined the mechanism of this inhibition in BL41 Burkitt lymphoma cells. PAO-dependent inhibition is not restricted to the BCR-mediated functions, as evidenced by the failure of the same cells to mobilize Ca2+ in response to CD19 cross-linking. In contrast, calcium response induced by a putative syk activator, H2O2, exhibited only a moderate sensitivity to PAO, demonstrating that PAO did not cause general suppression of all the functions leading to Ca2+ mobilization. BCR cross-linking or H2O2 treatment leads to the induction of almost complete non-responsiveness for the reciprocal stimulation. Since BCR cross-linking did not generate non-responsiveness to H2O2 in the presence of PAO, and PAO-treated cells remained responsive to syk activation by H2O2, we suppose that PAO may inhibit BCR-mediated signal transduction events upstream of syk activation. This assumption was supported by additional data, indicating that PAO was able to modulate functions of at least 2 different protein tyrosine kinase enzymes involved in BCR-mediated signaling. PAO induced rapid and dose-dependent tyrosine phosphorylation of lyn and selectively inhibited BCR-mediated tyrosine phosphorylation of syk. The results presented in this paper demonstrate that PAO may provoke cellular desensitization process by alteration of the signal transducer functions of lyn and syk tyrosine kinase enzymes.

Fine specificity of a rabbit antibody interacting with human IgG Fc receptor-like molecules

A polyclonal rabbit antibody raised against an Fc receptor (FcR)-like membrane glycoprotein fraction of chronic leukaemic lymphocytes has previously been prepared and partially characterized. This antibody, called AbA, was found to precipitate a 70-kDa and a 45-kDa fraction of the detergent lysate of U937 cells and to inhibit ligand binding to Fc gamma R on the P388D1 murine macrophage cell line. In the present work we have characterised this antibody further. All Fc gamma RII-positive B lymphoblastoid cell lines, as well as resting human B lymphocytes, were positively stained with the AbA antibody. U937 cells were found to be negative, but after stimulation with phorbol ester (PMA), 50% of the cells became positive. AbA antibody did not react with human T cell lines or with the T + 0 cell subset of peripheral blood. Monocytes were also negative. On the other hand, AbA antibody exhibited a dose-dependent inhibition of antibody-mediated cytotoxic reaction (ADCC) of monocytes, while not affecting K cell-mediated ADCC. It had an inhibitory effect of EA rosette formation of B cells and stimulated U937 cells. Furthermore, it interacted with the soluble form of Fc gamma RII released by activated B lymphocytes, and--similarly to IgG--precipitated a 33 kDa fraction from the supernatant of B cells.