Distinct pathways of CD4 and CD8 cells induce rapid target DNA fragmentation.
When activated with either Con A, a CD3-specific mAb, or Ag-pulsed B lymphoma (LK35.2) cells, CD4 (Th1) clones quickly induce DNA fragmentation in target cells followed by release of 51Cr-labeled intracellular materials. Both activated CD4 clones and CD8 (CTL) cells fragment target DNA into electrophoretically identical "ladder" pattern made of approximately 200 bp. The effect of various metabolic inhibitors on the ability of CD4 and CD8 cells to induce target DNA fragmentation was studied. Little effect was observed with the DNA synthesis inhibitor, mitomycin C. The RNA synthesis inhibitor, actinomycin D, and the protein synthesis inhibitor, cycloheximide, strongly inhibited the ability of CD4 cells, but not CD8 cells, to induce target DNA fragmentation. In contrast, target DNA fragmentation by CD8 cells, but not by CD4 cells, was inhibited by cholera toxin. Although cyclosporin A inhibited CD4 cells to fragment target DNA during the early phase (90 min) of E:T interaction, this inhibition was not sustained in the later phase (210 min) of the assay. Zinc ions inhibited the ability of both CD4 and CD8 cells to fragment target DNA. Treatment of effectors and targets with these inhibitors, followed by washings, demonstrated that the action of these inhibitors on effector cells alone is sufficient to inhibit target DNA fragmentation. The strong correlation among these parameters of DNA fragmentation and Cr-release assays supports the hypothesis of programed cell death. Although distinct cytolytic pathways are used by CD4 and CD8 cells to kill targets, both pathways deliver a signal that activates endonuclease(s), fragments target DNA, causes Cr-release, and lyses target cells. Taken together with our previous studies, the present findings demonstrate that activated cytolytic CD4 clones do not use perforin, serine proteases, and TNF as mediators for resistant target DNA fragmentation.
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Department of Medicine, Boston University School of Medicine, MA 02118.
D000818:Animals; D015496:CD4-Positive T-Lymphocytes / Q000502:physiology*; D002470:Cell Survival; D003513:Cycloheximide / Q000494:pharmacology; D003524:Cyclosporins / Q000494:pharmacology; D004247:DNA / Q000378:metabolism*; D003609:Dactinomycin / Q000494:pharmacology; D051379:Mice; D050378:T-Lymphocytes, Regulatory / Q000502:physiology*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D015032:Zinc / Q000494:pharmacology
D003524:Cyclosporins; D014409:Tumor Necrosis Factor-alpha; D003609:Dactinomycin; D004247:DNA; D003513:Cycloheximide; D015032:Zinc