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CD3ζ-chain expression is regulated by tumor necrosis factor via Src-like adaptor protein dependent proteosomal degradation in human T lymphocytes
  1. Barbara Érsek1,
  2. Viktor Molnár1,
  3. Andrea Balogh2,
  4. János Matkó2,
  5. Andrew P Cope3,
  6. Edit I Buzás1,
  7. András Falus1,
  8. György Nagy1
  1. 1Department of Genetics, Cell and Immunobiology, Semmelweis University, Medical School, Budapest, Hungary
  2. 2Eötvös Lóránd University, Department of Immunology, Budapest, Hungary
  3. 3Academic Department of Rheumatology, Centre for Molecular and Cellular Biology of Inflammation, Division of Immunology, Infection and Inflammatory Disease, King's College School of Medicine, King's College London, UK
  4. 4Department of Rheumatology, Semmelweis University, Medical School, Budapest, Hungary

Abstract

Background and objectives decreased expression of the T cell receptor (TCR) ζ-chain has been reported in several autoimmune, inflammatory and malignant diseases, suggesting that ζ-chain downregulation is common at sites of chronic inflammation. While ζ-chain is critically important in T lymphocyte activation, the mechanism of the decreased ζ-chain expression is not known. Src-like adaptor protein (SLAP) is a master regulator of T cell activation; previous data indicated that SLAP regulates immunoreceptor signaling. The authors have examined the mechanism and the functional consequences of CD3 ζ-chain downregulation.

Materials and methods CD3ζ and SLAP protein levels of Jurkat cells and primary human T lymphocytes were measured by Western blot. Jurkat cells were transiently transfected with siRNAs to silence SLAP, knockdown efficiency of the siRNAs was measured by real-time RT-PCR and by Western blot. For confocal microscopy experiments cells were transfected with eGFP-SLAP expression-ready, full-length cDNA vector or control eGFP vector. The colocalisation between CD3ζ and SLAP were determined by laser confocal microscopy. CD3ζ mRNA was measured by quantitative real-time RT-PCR, IL-2 level was measured by ELISA method.

Results TNF treatment of human T lymphocytes (15–40 ng/ml) selectively downregulates CD3 ζ-chain expression in a dose dependent manner (p<0.001), and decreases the activation induced IL-2 synthesis (p<0.01). Although blocking of the lysosomal compartment fails to restore the TNF-induced CD3 ζ-chain downregulation, the inhibition of the proteasome prevented the effect of TNF. Both SLAP expression and the colocalisation of SLAP with CD3 ζ-chain was enhanced by TNF treatment (p<0.05; p<0.009 respectively), while TNF induced ζ-chain downregulation was inhibited by silencing SLAP with siRNA (p<0.01).

Conclusions Our present data suggest that TNF modulates T cell activation during inflammatory processes, by regulating the amount of CD3 ζ-chain expression via SLAP dependent mechanism. These data indicate that SLAP dependent regulation of CD3 ζ-chain provides fine control of TCR signaling.

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