Franca Cofano

Activation of protein kinase C down-regulates IFN-gamma receptors

Treatment of mouse EL-4 cells with intracellular activators of protein kinase C, namely 4-phorbol 12-myristate 13-acetate (PMA) and diacylglycerol, resulted in 90% reduction in cell surface interferon-gamma (IFN-gamma) receptors as judged by iodinated-IFN-gamma binding. This did not seem to be due to a decreased in the receptor affinity, since that of the remaining surface receptors appeared to be significantly increased as shown in Scatchard plot analysis. Kinetics experiments revealed that a PMA treatment as short as 15 min was sufficient to induce a decrease of 30% of IFN-gamma receptors, whereas the highest levels of down-regulation were observed after 60-90 min. Treatment of EL-4 cells with calcium ionophore, A23187, although ineffective by itself, dramatically increased the ability of suboptimal PMA concentrations to mediate IFN-gamma receptor down-regulation. Finally, specificity studies revealed that PMA is particularly effective in decreasing the binding of IFN-gamma to T-lymphocytes. Altogether these results suggest a possible involvement of protein kinase C in the regulation of IFN-gamma receptor expression.

Monoclonal Antibodies against Mouse γ-Interferon Inhibit Tumoricidal Macrophage Activation by T Lymphocytes

A monoclonal antibody, AN-18.17.24, specific for murine interferon-gamma (IFN-gamma) was produced by immunizing Wistar rats with IFN-gamma secreted by a T-cell lymphoma, L12-R4, upon stimulation with phorbol myristic acetate (PMA). Antiviral activity as well as tumoricidal activation induced by PMA-stimulated L12-R4 cell supernatant or by Con A-stimulated normal spleen cells were neutralized at the same extent by AN-18 monoclonal antibody. Moreover, depletion experiments showed that inhibition of tumoricidal macrophage activation must be ascribed to the direct binding of the IFN-gamma molecule by AN-18 MAb and not to the interference of the monoclonal antibody with the cell surface IFN-gamma receptor. These studies conclusively demonstrate that in supernatants of T lymphocytes stimulated with polyclonal activators IFN-gamma was the only molecule responsible for macrophage activation in tumor cell killing.

Production of Antibodies Against the Murine IFN-γ Receptor

Stimulation of T-lymphocytes with antigens or mitogens triggers the release of several lymphokines, that regulate the immune response (1). Of these, IFN- γ has been shown to control the activity of several cell populations, namely T- and B-lymphocytes, natural killer cells and macrophages (2). The initial IFN-target cell interaction occurs at specific membrane receptors (3). Previous data have demonstrated that the number of binding sites varies from 2,000 to 25,000, with dissociation constants (Kd) between 10-9 and 10-11 (3). One of the major obstacles, however, to characterization and purification of the receptor molecule is that it is expressed in a number of binding sites per single cell insufficient for its purification and biochemical characterization.

Interferon-γ plays a crucial role in T lymphocyte reaction against alloantigens

SummaryIn this study we report that addition to mixed lymphocyte reactions of monoclonal antibodies to interferon-γ inhibits alloantigen recognition and induction of cytotoxic T lymphocytes by inducing early and highly effective suppressor T lymphocytes. This inhibitory activity is not confined toin vitro models, since daily local injection of these antibodies in CBA/J mice blocks the rejection of fully allogeneic tumor cells consistently displayed by untreated CBA/J mcie.