IgE receptor-mediated phosphatidylinositol hydrolysis was studied in rat basophilic leukemia cells permeabilized with alpha toxin from S. aureus. The rate of PI hydrolysis in permeabilized cells is twice that observed in intact cells. As we previously demonstrated for IgE receptor-mediated degranulation of RBL cells permeabilized with alpha toxin, receptor-mediated phosphatidylinositol hydrolysis in permeabilized cells proceeds if Ca2+ is omitted from the buffer. In addition, permeabilized cells are extremely sensitive to small concentrations of selected anions. For example, 1 mM glutamate causes a 2 fold enhancement of receptor-mediated phosphatidylinositol hydrolysis, while 2 mM sulfate inhibits the reaction by greater than 60%. Moreover, the relatively high concentrations of chloride often used by investigators when studying permeabilized cells are also inhibitory. Diisopropylfluorophosphate (DFP), an irreversible inhibitor of serine esterases, inhibits IgE receptor-mediated phosphatidylinositol hydrolysis in a dose-.dependent manner with 50% inhibition occurring at 3 mM DFP. DFP must be present when the cells are triggered for it to have an inhibitory effect. Therefore, if cells are incubated with DFP, washed, and triggered in the absence of DFP, there is no inhibition. In addition to inhibition of receptor-mediated phosphatidylinositol hydrolysis in intact cells, incubation of RBL cells with DFP results in inhibition of phospholipase C activity when the treated cells are used to make a cytosolic extract and the phospholipase C activity is measured. Moreover, studies performed on purified phospholipase C demonstrate that this enzyme is directly inhibited by the presence of DFP in the assay. These results suggest that DFP is able to bind to and inhibit phospholipase C. This finding is consistent with all of the data concerning inhibition of mast cell activation by DFP and could explain the effect of this inhibitor on IgE receptor-mediated exocytosis. Z01AI00494 Immunity to infection by the intracellular parasite Leishmania is mediated by sensitized T cells, however direct recognition of infected macrophages by T cells has not been clearly demonstrated. Experiments addressing this question have been confounded by the possibility that T cell activation in these systems might be due to release of Leishmania antigens by infected cells and subsequently presented by contaminating populations of uninfected antigen presenting cells (APCs such as B cells or uninfected macrophages. We have addressed this issue using peripheral blood mononuclear cells (PBMNCs) from patients with healing cutaneous L. major infection. Monocytes were purified from PBMNCs and then infected with serum opsonized L. major or L. donovani amastigotes. Cells were then lightly fixed to stop active infection and prevent further processing and possible release of parasite antigens. Addition of functionally pure autologous T cells resulted in strong proliferative responses to both L. major, and L. donovani fixed infected monocytes. This stimulation could not be augmented by the addition of uninfected monocytes. Supernatents from infected monocytes failed to stimulate unfractionated PBMNCs indicating the absence of non-celi associated soluble antigens. We conclude that Leishmania infected macrophages are able to efficiently process and present antigens to sensitized T cells. Identification of the antigens which are being recognized by T cells from immune individuals is continuing using previously generated T cell clones. Leishmania antigens separated by 2-D electrophoresis as well as FPLC and phenyl-sepharose chromatography have been used to identify specific proteins which activate a number of these clones. The development of DNA probes for cytokines produced by activated lymphocytes and macrophages has been completed and they will now be used to reexamine the responses of lymphocytes from patients with cutaneous, mucocutaneous or visceral disease to defined Leishmania antigens or to autologous infected macrophages.
