Physiologic signaling in normal human T-cells

mRNA phenotyping by northern blot analysis and reverse transcription-polymerase chain reaction

Kenneth J. Wieder, Gerd Walz, Bernd Zanker, Prabodh Sehajpal, Vijay K. Sharma, Edward Skolnik, Terry B. Strom, Manikkam Suthanthiran

Research output: Contribution to journalArticle

8 Citations (Scopus)

Abstract

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-γ (IFN-γ), 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-γ, 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-γ gene expression in accessory cell-depleted T-cells stimulated by crosslinking 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.

Original languageEnglish
Pages (from-to)41-51
Number of pages11
JournalCellular Immunology
Volume128
Issue number1
DOIs
Publication statusPublished - 1 Jan 1990
Externally publishedYes

Fingerprint

Northern Blotting
Reverse Transcription
Ionomycin
Interleukin-2
T-Lymphocytes
Polymerase Chain Reaction
Messenger RNA
CD2 Antigens
Interferons
CD3 Antigens
Gene Expression
Transcriptional Activation
ras Genes
Calcium Ionophores
Lymphokines
Protein Kinase C
Cyclosporine
Genes
1,2-dioctanoylglycerol
Calcium

ASJC Scopus subject areas

  • Immunology

Cite this

Physiologic signaling in normal human T-cells : mRNA phenotyping by northern blot analysis and reverse transcription-polymerase chain reaction. / Wieder, Kenneth J.; Walz, Gerd; Zanker, Bernd; Sehajpal, Prabodh; Sharma, Vijay K.; Skolnik, Edward; Strom, Terry B.; Suthanthiran, Manikkam.

In: Cellular Immunology, Vol. 128, No. 1, 01.01.1990, p. 41-51.

Research output: Contribution to journalArticle

Wieder, Kenneth J. ; Walz, Gerd ; Zanker, Bernd ; Sehajpal, Prabodh ; Sharma, Vijay K. ; Skolnik, Edward ; Strom, Terry B. ; Suthanthiran, Manikkam. / Physiologic signaling in normal human T-cells : mRNA phenotyping by northern blot analysis and reverse transcription-polymerase chain reaction. In: Cellular Immunology. 1990 ; Vol. 128, No. 1. pp. 41-51.
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