Physiologic signaling in normal human T-cells: mRNA phenotyping by northern blot analysis and reverse transcription-polymerase chain reaction
| Autor: | Gerd Walz, Bernd Zanker, Manikkam Suthanthiran, Edward Skolnik, Kenneth J. Wieder, Vijay K. Sharma, Terry B. Strom, Prabodh K. Sehajpal |
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| Rok vydání: | 1990 |
| Předmět: |
Antigens
Differentiation T-Lymphocyte T cell T-Lymphocytes Immunology Molecular Sequence Data CD2 Antigens Oligonucleotides Gene Expression Cyclosporins Biology In Vitro Techniques Polymerase Chain Reaction Diglycerides Proto-Oncogene Proteins p21(ras) chemistry.chemical_compound Interferon-gamma Transcription (biology) Proto-Oncogene Proteins Gene expression medicine Humans Northern blot RNA Messenger Receptors Immunologic Messenger RNA Base Sequence Activator (genetics) Ionomycin Blotting Northern Molecular biology Reverse transcription polymerase chain reaction medicine.anatomical_structure chemistry Interleukin-2 Signal Transduction |
| Zdroj: | Cellular immunology. 128(1) |
| ISSN: | 0008-8749 |
| Popis: | Synergy between ionomycin and sn -1,2-dioctanoylglycerol (diC 8 ) 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 diC 8 , 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 diC 8 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 diC 8 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, diC 8 , 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. |
| Databáze: | OpenAIRE |
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