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How does tumour necrosis factor uncouple T-cell receptor-induced IL-2 gene expression in murine T cells?
Arthritis Research & Therapyvolume 7, Article number: P41 (2005)
T-cell receptor (TCR)-induced IL-2 expression is suppressed at the level of mRNA and protein following chronic culture of T cells with tumour necrosis factor (TNF) at picomolar concentrations, an effect that is reversible upon removal of TNF. This TCR hyporesponsiveness is reminiscent of that of T cells recovered from inflamed sites such as the rheumatoid synovium. We have found that, although chronic TNF attenuates TCRζ expression and TCR-proximal signalling, IL-2 expression is suppressed regardless of whether cells are stimulated via the TCR, or with phorbol ester and calcium ionophore to activate the MAPK and NFAT pathways directly. Suppression therefore occurs independently of TCR-proximal effects of TNF [1, 2].
We are studying the mechanisms by which chronic TNF suppresses T-cell responses in the CD4+ mouse T-cell hybridoma clone, 11A2. We have compared the stability of induced IL-2 mRNA, and the regulation of transcription factors important for IL-2 gene expression, in control and TNF-treated cells; and we have recently begun to study effects of chronic TNF on chromatin remodelling across the IL-2 proximal promoter (pIL-2), which precedes initiation of transcription, using the chromatin accessibility real-time PCR (CHART-PCR) assay .
Cells were grown in the presence or absence of 2.5 ng/ml murine TNF for 8 days. Washed cells were stimulated with either plate-bound anti-CD3ε, or phorbol 12-myristate 13-acetate (PMA) and ionomycin, for 4 hours.
mRNA stability assay
Actinomycin D was added and cells harvested over a further 4-hour time-course. Total RNA was extracted and probed for IL-2 mRNA by ribonuclease protection assay.
Transcription factor regulation
Whole cell lysates were probed for NF-κB and IκB family proteins by immunoblot.
Nuclei were isolated and subjected to limited nuclease digestion. DNA was extracted and quantitative analysis of target sequenceswithin pIL-2 carried out by real-time PCR. An increase in the threshold cycle (CT) of a target sequence PCR product from activated cells was indicative of stimulation-induced chromatin remodelling.
IL-2 mRNA induced via TCR was unstable (t1/2 < 30 min), whereas PMA and ionomycin stabilised IL-2 mRNA strongly (t1/2 > 2 hours). However, the initial rate of decay was similar in control and TNF-treated cells, suggesting that reduced expression in TNF-treated cells was not due to decreased stability of IL-2 mRNA. c-Rel, IκBβ and IκBε were regulated differently in control and TNF-treated cells: expression of IκBε was comparatively enhanced, while that of c-Rel and IκBβ was attenuated in TNF-treated cells. These altered responses may affect the ability of TNF-treated cells to remodel pIL-2 productively through NFκB consensus binding upon cell stimulation . This hypothesis is now being tested by CHART-PCR.
Chronic culture of murine T cells in TNF does not alter the stability of IL-2 mRNA induced via TCR, or by PMA and ionomycin, in those cells. However, altered regulation of NF-κB expression and activity in TNF-treated cells may contribute to poor inducibility of IL-2 through effects on stimulus-induced changes in chromatin conformation across pIL-2.
Isomäki P, Panesar M, Annenkov A, Clark JM, Foxwell BM, Chernajovsky Y, Cope AP: Prolonged exposure of T cells to TNF down-regulates TCR zeta and expression of the TCR/CD3 complex at the cell surface. J Immunol. 2001, 166: 5495-5507.
Clark JM, Annenkov AE, Panesar M, Isomäki P, Chernajovsky Y, Cope AP: T cell receptor zeta reconstitution fails to restore responses of T cells rendered hyporesponsive by tumor necrosis factor alpha. Proc Natl Acad Sci USA. 2004, 101: 1696-1701. 10.1073/pnas.0308231100.
Rao S, Procko E, Shannon MF: Chromatin remodeling, measured by a novel real-time polymerase chain reaction assay, across the proximal promoter region of the IL-2 gene. J Immunol. 2001, 167: 4494-4503.
Rao S, Gerondakis S, Woltring D, Shannon MF: c-Rel is required for chromatin remodeling across the IL-2 gene promoter. J Immunol. 2003, 170: 3724-3731.