cytochalasin-d and Leukemia--Basophilic--Acute

Cross-linking of FcepsilonRI on rat basophilic leukemia (RBL) cells initiates a signaling cascade leading to degranulation of the cells and the release of inflammatory mediators. Inhibitors that disrupt microfilaments, such as latrunculin and cytochalasin D, do not cause any degranulation on their own, but they do enhance FcepsilonRI-mediated degranulation. Dose-response studies show a good correlation between inhibition of actin polymerization and increased degranulation. In RBL cells, latrunculin causes a decrease in basal levels of filamentous actin (F-actin), while cytochalasin D does not. This is particularly evident in the Triton-insoluble pool of F-actin which is highly cross-linked and associated with the plasma membrane. A concentration of 500 nM latrunculin decreases the basal level of Triton-insoluble F-actin by 60-70% and total F-actin levels by 25%. Latrunculin increases both the rate and extent of Ag-induced degranulation while having no effect on pervanadate-induced degranulation. Pervanadate activates the signaling pathways directly and bypasses the cross-linking of the receptor. RBL cells, activated through FcepsilonRI in the presence of latrunculin, show increased phospholipase activity as well as increased tyrosine phosphorylation of Syk and increased tyrosine phosphorylation of the receptor itself by the tyrosine kinase Lyn. This indicates that the very earliest signaling events after receptor cross-linking are enhanced. These results suggest that actin microfilaments may interact, either directly or indirectly, with the receptor itself and that they may regulate the signaling process at the level of receptor phosphorylation. Microfilaments may possibly act by uncoupling Lyn from the cross-linked receptor.

Topics: Actin Cytoskeleton; Actins; Animals; Bridged Bicyclo Compounds, Heterocyclic; Cell Degranulation; Cytochalasin D; Dinitrophenols; Dose-Response Relationship, Immunologic; Down-Regulation; Haptens; Leukemia, Basophilic, Acute; Marine Toxins; Mast Cells; Porifera; Rats; Receptors, IgE; Serum Albumin, Bovine; Signal Transduction; Thiazoles; Thiazolidines; Tumor Cells, Cultured

The mast cell lines rat basophilic leukemia (RBL) and mouse C57 cells respond to IgE/antigen complexes by degranulation. Treatment of these cells with 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), (10-100 nM) for 24-48 h enhanced IgE/antigen-induced exocytosis as monitored by release of hexosaminidase. A short term incubation with the hormone did not affect exocytosis, ruling out a rapid non genomic mechanism. The presence of vitamin D receptors, demonstrated by immunoblotting and the lack of effect of 24,25(OH)2D3 suggest a role for these receptors in the enhancing effect. 1,25(OH)2D3 also enhanced exocytosis induced by the calcium ionophore A23187 in the presence or absence of phorbol ester indicating modulation of events distal to signal transduction. 1,25(OH)2D3 enhanced exocytosis in the presence of cytochalasin D, indicating that the action of the hormone is not due to effects on microfilament structure. The results of this study suggest that 1,25(OH)2D3 may affect the allergic or pro-inflammatory potential of mast cells.

Topics: Animals; Antigens; beta-N-Acetylhexosaminidases; Calcimycin; Calcitriol; Cell Degranulation; Cytochalasin D; Dinitrophenols; Exocytosis; Immunoglobulin E; Leukemia, Basophilic, Acute; Mast Cells; Mice; Nucleic Acid Synthesis Inhibitors; Rats; Receptors, Calcitriol; Tumor Cells, Cultured

Phorbol myristate acetate (PMA) can induce a rapid and significant decrease in the expression of IgE receptors on RBL-2H3 cells. Fluorescence microscopy confirmed that the down-regulation is due to internalization of receptors. The endocytotic response to PMA shares several characteristics with endocytosis induced by immunochemical aggregation of surface-bound monomeric IgE: the rates of internalization both have a t1/2 of about 5 min, a maximum of 35% of the surface-bound IgE can be endocytosed by the action of PMA (50% by receptor aggregation), endocytosis is sustained for at least up to 60 min, neither stimulus requires extracellular Ca2+ and endocytosis induced by either stimulus is an active process, i.e., is dependent on temperature and cellular energy. Biochemical studies revealed some differences between the endocytotic responses to the two stimuli. After prolonged treatment of cells with dexamethasone, only endocytosis induced by PMA is inhibited. Cells depleted of protein kinase C by prolonged exposure to PMA can sustain a significant endocytotic response to aggregation of IgE receptors, but become completely desensitized to PMA. These data suggest that different biochemical pathways mediate the signals from the two stimuli and that protein kinase C is directly involved in endocytosis induced by PMA but does not have a major role in endocytosis induced by receptor aggregation.

Topics: Animals; Antigens, Differentiation, B-Lymphocyte; Cytochalasin D; Diglycerides; Endocytosis; Immunoglobulin E; Leukemia, Basophilic, Acute; Protein Kinase C; Rats; Receptors, Fc; Receptors, IgE; Tetradecanoylphorbol Acetate