Physiologic signaling in normal human T-cells: mRNA phenotyping by northern blot analysis and reverse transcription-polymerase chain reaction.
Synergy between ionomycin and sn-1,2-dioctanoylglycerol (diC8) was shown at the level of lymphokine gene transcription. Transcriptional activation of interleukin-2 (IL-2), interferon-gamma (IFN-gamma), and the protooncogene H-ras was accomplished by signaling highly purified normal human resting T-lymphocytes (T-cells) with diC8, a physiologic regulator of protein kinase C, and the calcium ionophore, ionomycin. Northern blot analysis of mRNA for early T-cell activation genes demonstrated the synergism between diC8 and ionomycin at the gene induction level. To amplify very low levels of IL-2 mRNA, sequential reverse transcription and polymerase chain reaction (RT-PCR) of T cell mRNA were used to demonstrate the capacity of the calcium signal (ionomycin) to promote low-level IL-2 transcription in normal human T-cells without additional signals. Cyclosporine (CsA) prevented diC8 and ionomycin-induced expression of IL-2, IFN-gamma, and H-ras genes. The completeness of its inhibitory effect was evident by the absence of IL-2 transcripts in CsA-treated cultures screened by the RT-PCR technique. CsA also prevented IL-2 and IFN-gamma gene expression in accessory cell-depleted T-cells stimulated by cross-linking the CD2 and CD3 antigens on the cell surface. Our observations demonstrate that a physiologic regulator of PKC, diC8, and cell surface crosslinking of the CD2 and CD3 antigen, promote gene expression in normal human quiescent T-cells independently of accessory cells, and that CsA prevents gene expression via a direct effect on T-cells.
Wieder
K J
KJ
Beth Israel Hospital, Harvard Medical School, Boston, Massachusetts 02215.
Walz
G
G
Zanker
B
B
Sehajpal
P
P
Sharma
V K
VK
Skolnik
E
E
Strom
T B
TB
Suthanthiran
M
M
D000945:Antigens, Differentiation, T-Lymphocyte / Q000502:physiology; D001483:Base Sequence; D015152:Blotting, Northern; D018801:CD2 Antigens; D003524:Cyclosporins / Q000494:pharmacology; D004075:Diglycerides / Q000494:pharmacology; D015870:Gene Expression / Q000187:drug effects; D006801:Humans; D066298:In Vitro Techniques; D007371:Interferon-gamma / Q000235:genetics; D007376:Interleukin-2 / Q000235:genetics; D015759:Ionomycin / Q000494:pharmacology; D008969:Molecular Sequence Data; D009841:Oligonucleotides; D016133:Polymerase Chain Reaction; D011518:Proto-Oncogene Proteins / Q000235:genetics; D016283:Proto-Oncogene Proteins p21(ras); D012333:RNA, Messenger / Q000235:genetics; D011971:Receptors, Immunologic / Q000502:physiology; D015398:Signal Transduction / Q000187:drug effects; D013601:T-Lymphocytes / Q000502:physiology*
D000945:Antigens, Differentiation, T-Lymphocyte; D018801:CD2 Antigens; D003524:Cyclosporins; D004075:Diglycerides; D007376:Interleukin-2; D009841:Oligonucleotides; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger; D011971:Receptors, Immunologic; C044295:1,2-dioctanoylglycerol; D015759:Ionomycin; D007371:Interferon-gamma; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras)