herbimycin and Mast-Cell-Sarcoma

This study describes a form of partial agonism for a CD8+ CTL clone, S15, in which perforin-dependent killing and IFN-gamma production were lost but Fas (APO1 or CD95)-dependent cytotoxicity preserved. Cloned S15 CTL are H-2Kd restricted and specific for a photoreactive derivative of the Plasmodium berghei circumsporozoite peptide PbCS 252-260 (SYIPSAEKI). The presence of a photoactivatable group in the epitope permitted assessment of TCR-ligand binding by TCR photoaffinity labeling. Selective activation of Fas-dependent killing was observed for a peptide-derivative variant containing a modified photoreactive group. A similar functional response was obtained after binding of the wild-type peptide derivative upon blocking of CD8 participation in TCR-ligand binding. The epitope modification or blocking of CD8 resulted in an > or = 8-fold decrease in TCR-ligand binding. In both cases, phosphorylation of zeta-chain and ZAP-70, as well as calcium mobilization were reduced close to background levels, indicating that activation of Fas-dependent cytotoxicity required weaker TCR signaling than activation of perforin-dependent killing or IFN-gamma production. Consistent with this, we observed that depletion of the protein tyrosine kinase p56(lck) by preincubation of S15 CTL with herbimycin A severely impaired perforin- but not Fas-dependent cytotoxicity. Together with the observation that S15 CTL constitutively express Fas ligand, these results indicate that TCR signaling too weak to elicit perforin-dependent cytotoxicity or cytokine production can induce Fas-dependent cytotoxicity, possibly by translocation of preformed Fas ligand to the cell surface.

Topics: Animals; Antibodies, Monoclonal; Azides; Benzoquinones; Calcium Signaling; CD8 Antigens; Clone Cells; Cytotoxicity, Immunologic; Enzyme Inhibitors; Epitopes; Fas Ligand Protein; fas Receptor; H-2 Antigens; Immunoglobulin Fab Fragments; Interferon-gamma; Lactams, Macrocyclic; Lymphocyte Activation; Lymphocyte Specific Protein Tyrosine Kinase p56(lck); Mast-Cell Sarcoma; Membrane Glycoproteins; Membrane Proteins; Mice; Peptide Fragments; Perforin; Phosphorylation; Photoaffinity Labels; Plasmodium berghei; Pore Forming Cytotoxic Proteins; Protein Processing, Post-Translational; Protein-Tyrosine Kinases; Protozoan Proteins; Quinones; Receptors, Antigen, T-Cell; Rifabutin; Salicylates; T-Lymphocytes, Cytotoxic; ZAP-70 Protein-Tyrosine Kinase

Mast cells, mastocytoma cells and basophil leukaemia cells are well-established producers of leukotrienes when grown and stimulated appropriately. I report that the cells' ability to produce leukotrienes is dependent on the cells' proliferative status or their provision with growth factors. Proliferating MC/9 and subconfluent RBL2H3 cells respond maximally to stimulation by 1 microM ionomycin with the production of 56 and 32 pmol of cysteinyl-leukotrienes/10(6) cells respectively. In contrast, confluent RBL2H3 or growth-arrested MC/9 cells lose their ability to generate leukotrienes in response to ionomycin treatment. This rapid down-regulation of leukotriene synthesis is also observed when proliferating RBL2H3 cells are transferred to growth-factor-free medium, wherein cellular leukotriene-synthesis capacity has an apparent half-lifetime of 60 min. Transfer back into growth medium results in the regeneration of leukotriene synthesis capacity within 6 h. In growth-arrested MC/9 cells, leukotriene production ability can at least partially be restored by priming the cells with interleukin 3, but not with interleukin 4. In RBL2H3 cells, pretreatment with protein tyrosine kinase inhibitors such as genistein (5 min, 37 microM), herbimycin A (6 h, 3 microM) or tyrphostin 25 (16 h, 100 microM) completely inhibits leukotriene generation, whereas okadaic acid (15 min, 0.5 microM) has no effect. Under these conditions, both genistein and herbimycin A strongly impair ionomycin-induced protein tyrosine phosphorylation. Our study indicates that leukotriene generation in these tumour cells is tightly regulated by their proliferation status and supply with growth factors, and cell stimulation towards leukotriene synthesis appears to involve protein tyrosine kinase activity.

Topics: Animals; Benzoquinones; Catechols; Cell Division; Cell Line; Genistein; Interleukin-3; Interleukin-4; Ionomycin; Isoflavones; Kinetics; Lactams, Macrocyclic; Leukemia, Basophilic, Acute; Leukotrienes; Mast-Cell Sarcoma; Mice; Nitriles; Phosphoproteins; Phosphotyrosine; Protein-Tyrosine Kinases; Quinones; Rats; Rifabutin; Tumor Cells, Cultured; Tyrosine; Tyrphostins