Role of reactive oxygen intermediates in TCR-induced death of T cell blasts and hybridomas.
The functional role of reactive oxygen intermediates (ROI) in activation-induced death of mature T lymphocytes and hybridomas was tested using antioxidants and inhibitors of enzymes that generate oxidants. These agents were shown to inhibit TCR-triggered death in a concentration-dependent manner, suggesting a possible role of ROI in this death. Since the TCR-induced death of both human T blasts and the murine T cell hybridoma 2B4 involve an initial step of TCR-induced Fas ligand (FasL) up-regulation followed by a lethal step induced by Fas cross-linking, both steps were examined separately for ROI dependence. The thiol antioxidants N-acetyl cysteine and glutathione blocked Fas-induced death triggered via cross-linking either by IgM anti-Fas or cell-bound FasL, while the other inhibitors of activation-induced death did not block this late lethal step. None of the agents used blocked early events after TCR ligation, as seen by the lack of inhibition of IL-2 secretion or CD69 up-regulation. However, the nonthiol agents that blocked activation-induced death all blocked FasL up-regulation induced by TCR signals in the hybridoma, as measured by a functional assay. Agents inhibiting FasL up-regulation also inhibited activation-induced ROI generation in the hybridoma, as detected by flow cytometry using dihydrorhodamine oxidation. Furthermore, a good correlation was found between the extent of ROI generation and functional FasL expression in 2B4 cells. Thus, while ROI do not appear to act as downstream mediators of apoptotic death induced by steroid or Fas cross-linking in T cells, they are generated by TCR signaling and appear to participate in FasL up-regulation.
Williams
M S
MS
Experimental Immunology Branch, National Cancer Institute, Bethesda, MD 20892, USA.
Henkart
P A
PA
D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D016923:Cell Death / Q000187:drug effects; D053222:Fas Ligand Protein; D006801:Humans; D006825:Hybridomas / Q000187:drug effects / Q000378:metabolism*; D008024:Ligands; D008213:Lymphocyte Activation / Q000187:drug effects; D008562:Membrane Glycoproteins / Q000096:biosynthesis; D051379:Mice; D010100:Oxygen / Q000037:antagonists & inhibitors; D017382:Reactive Oxygen Species / Q000378:metabolism / Q000502:physiology*; D011948:Receptors, Antigen, T-Cell / Q000187:drug effects / Q000502:physiology*; D013438:Sulfhydryl Compounds / Q000494:pharmacology; D013601:T-Lymphocytes / Q000187:drug effects / Q000378:metabolism*; D019014:fas Receptor / Q000187:drug effects
D000975:Antioxidants; C505661:FASLG protein, human; D053222:Fas Ligand Protein; C505658:Fasl protein, mouse; D008024:Ligands; D008562:Membrane Glycoproteins; D017382:Reactive Oxygen Species; D011948:Receptors, Antigen, T-Cell; D013438:Sulfhydryl Compounds; D019014:fas Receptor; D010100:Oxygen