Background and Objectives although ζ-chain downregulation of human T lymhocytes is common at sites of chronic inflammation, the precise mechanism of ζ-chain regulation is not known. Src-like adaptor protein (SLAP) is a regulator of T cell activation; earlier data have reported that SLAP regulates immunoreceptor signalling. We studied the mechanism of CD3 ζ-chain downregulation.
Materials and Methods CD3ζ and SLAP protein levels of 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 cDNA vector or control eGFP vector. The colocalisation between CD3ζ and SLAP were measured by laser confocal microscopy. CD3ζ mRNA was measured by quantitative real-time RT-PCR, IL-2 level was measured by ELISA.
Results in vitro TNF treatment of human T cells selectively, dose dependently and reversibly downregulates CD3 ζ-chain expression and inhibits activation-induced IL-2 expression (p < 0.01). Inhibition of the proteasome prevented the effect of TNF on CD3 ζ-chain expression. The colocalization of SLAP with CD3 ζ-chain and the SLAP expression were enhanced by TNF treatment (p < 0.01 and p < 0.05, respectively). SLAP silencing with small interfering RNA inhibited the TNF-induced ζ-chain downregulation. SLAP levels of the CD4 T cells, isolated from patients with rheumatoid arthritis were higher than that of the healthy donors’ (p < 0.05). In addition, in vitro TNF treatment did not alter the SLAP expression of the CD4 lymphocytes of anti-TNF therapy-treated RA patients.
Conclusions our present data suggest that TNF regulates T cell activation during inflammatory processes, by altering CD3 ζ-chain expression via a SLAP-dependent mechanism. Thereafter SLAP-dependent regulation of CD3 ζ-chain may have an important role in the fine control of TCR signalling during chronic inflammation. SLAP may have a role in the pathomechanism of RA.
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