The molecular mechanism of FasL-mediated cytotoxicity by CD4+ Th1 clones.
Murine CD4+ Th1 clones require de novo synthesis of proteins to express a cytotoxicity that is mediated by de novo synthesized Fas ligand (FasL). The cytotoxic process of the CD4+ Th1 effectors can be separated into four stages, namely conjugate formation, activation, lethal hit, and effector-independent target cell death. The present study describes the cytotoxic process in terms of FasL induction and Fas/FasL molecular interactions for death signal transduction. Fas-Ig fusion proteins, cycloheximide, actinomycin D, and EGTA+MgCl2 were used to analyze each stage of the cytotoxic process in terms of FasL/Fas participation. The results demonstrate that the activation-induced de novo mRNA and protein synthesis were for FasL, which provided the predominant cytotoxic activity of CD4+ Th1 effectors. Once activated, Th1 effectors express cytotoxic activity in the presence of EGTA+MgCl2, an experimental [Ca2+]ext-independent condition characteristic of FasL-mediated cytotoxicity. The ability of Fas-Ig to inhibit target lysis declined rapidly after conjugate formation, indicating that FasL-mediated lethal hit is critically dependent on conjugate formation and, once delivered, the effector-independent target lysis proceeds. After the lethal hit stage, transduction of Fas-mediated death signal was independent of de novo synthesis of macro-molecules in targets because treatments that inhibited more than 98% of the macromolecule synthesis had little effect on target lysis. Our study provides the first molecular view in terms of FasL/Fas of the cytotoxic process of CD+ Th1 cells.
el-Khatib
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Department of Pathology, Boston University Medical Center, Massachusetts 02118, USA.
Stanger
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BZ
Dogan
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Cui
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Ju
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D000818:Animals; D000954:Antigens, Surface / Q000502:physiology; D001483:Base Sequence; D015704:CD4 Antigens / Q000032:analysis*; D000001:Calcimycin / Q000494:pharmacology; D002999:Clone Cells; D003513:Cycloheximide / Q000494:pharmacology; D003602:Cytotoxicity, Immunologic / Q000187:drug effects / Q000276:immunology*; D004533:Egtazic Acid / Q000494:pharmacology; D053222:Fas Ligand Protein; D007136:Immunoglobulins / Q000502:physiology; D008213:Lymphocyte Activation / Q000276:immunology; D015636:Magnesium Chloride / Q000494:pharmacology; D008562:Membrane Glycoproteins / Q000096:biosynthesis / Q000502:physiology*; D051379:Mice; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000502:physiology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D018417:Th1 Cells / Q000276:immunology* / Q000502:physiology*; D019014:fas Receptor
D000954:Antigens, Surface; D015704:CD4 Antigens; D053222:Fas Ligand Protein; C505658:Fasl protein, mouse; D007136:Immunoglobulins; D008562:Membrane Glycoproteins; D011993:Recombinant Fusion Proteins; D019014:fas Receptor; D015636:Magnesium Chloride; D000001:Calcimycin; D004533:Egtazic Acid; D003513:Cycloheximide; D013755:Tetradecanoylphorbol Acetate