Simultaneous transcription of eleven cytokines in human alloreactive T lymphocyte clones after stimulation by phorbol ester and A23187.
Human alloreactive T lymphocyte clones derived from cells invading a rejected kidney allograft, were analyzed for their ability to transcribe eleven cytokine genes under phorbol ester (PMA) plus calcium ionophore (CaI A 23187) stimulation. In addition to the positive signal previously obtained for IL-2 transcripts, strong specific patterns were seen with cytoplasmic dot hybridizations for IFN gamma and GM-CSF mRNAs in all the 17 clones screened. For the remaining transcripts (IL-3, IL-4, IL-5, IL-6, TNF alpha, LT, M-CSF and HILDA/LIF), these techniques proved to be inadequate. Northern-blots were therefore performed on three clones exhibiting different phenotypes (CD4+ CD8- non cytotoxic, CD4+ CD8- cytotoxic and CD4- CD8+ cytotoxic). Positive specific signal with the eleven probes could be obtained. Nevertheless, the IL-6 message was found only in the helper clone and the TNF alpha transcript appeared at a later time point compared to the other cytokine messages (its maximum expression was observed around 24 hours post-stimulation). In conclusion, we demonstrate that under PMA+CaI activation, one clone is able to simultaneously transcribe at least eleven lymphokine genes. Except, perhaps for IL-6, the pattern of lymphokine transcription did not permit us to distinguish between different lymphocyte subsets.
INSERM U.211, Institut de biologie, Nantes, France.
M M
MM
Hallet
M A
MA
Peyrat
J P
JP
Soulillou
J F
JF
Moreau
D015152:Blotting, Northern; D015496:CD4-Positive T-Lymphocytes / Q000187:drug effects / Q000378:metabolism; D000001:Calcimycin / Q000494:pharmacology*; D016207:Cytokines / Q000096:biosynthesis*; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D008213:Lymphocyte Activation / Q000187:drug effects; D008222:Lymphokines / Q000096:biosynthesis; D012333:RNA, Messenger / Q000032:analysis; D013268:Stimulation, Chemical; D016176:T-Lymphocyte Subsets / Q000187:drug effects* / Q000378:metabolism; D013602:T-Lymphocytes, Cytotoxic / Q000187:drug effects / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D014158:Transcription, Genetic / Q000187:drug effects*
D016207:Cytokines; D008222:Lymphokines; D012333:RNA, Messenger; D000001:Calcimycin; D013755:Tetradecanoylphorbol Acetate