TNFR1-induced sphingomyelinase activation modulates TCR signaling by impairing store-operated Ca2+ influx

Abstract

Tumor necrosis factor alpha (TNF-alpha) is a potent, pleiotrophic cytokine, which is proinflammatory but can also suppress T lymphocyte function. In chronic inflammatory disease such as rheumatoid arthritis, exposure of T cells to TNF-alpha alters their ability to mount a response by modulating the T cell receptor (TCR) signaling pathway, but the mechanisms involved remain obscure. Here, we investigated the specific role of TNF receptor 1 (TNFR1) signaling in the modulation of the TCR signaling pathway. We observed a down-regulation of the intracellular calcium ([Ca(2+)](i)) signal in Jurkat T cells after just 30 min exposure to TNF-alpha, and maximum suppression was reached after 3 h. This effect was transient, and signals returned to normal after 12 h. This depression of [Ca(2+)](i) was also observed in human CD4+ T lymphocytes. The change in Ca(2+) signal was related to a decrease in the plasma membrane Ca(2+) influx, which was apparent even when the TCR signal was bypassed using thapsigargin to induce a Ca(2+) influx. The role of TNF-alpha-induced activation of the sphingolipid cascade in this pathway was examined. The engagement of TNFR1 by TNF-alpha led to a time-dependent increase in acid sphingomyelinase (SMase; ASM) activity, corresponding with a decrease in cellular sphingomyelin. In parallel, there was an increase in cellular ceramide, which correlated directly with the decrease in the magnitude of the Ca(2+) response to phytohemagglutinin. Exogenous addition of SMase or ceramide mimicked the effects of TNFR1 signals on Ca(2+) responses in Jurkat T cells. Direct evidence for the activation of ASM in this pathway was provided by complete abrogation of the TNF-alpha-induced inhibition of the Ca(2+) influx in an ASM-deficient murine T cell line (OT-II(+/+)ASM(-/-)). This potent ability of TNF-alpha to rapidly modulate the TCR Ca(2+) signal via TNFR1-induced ASM activation can explain its suppressive effect on T cell function. This TNFR1 signaling pathway may play a role as an important regulator of T cell responses.

Elevated sphingomyelinase activity and ceramide concentration in serum of patients undergoing high dose spatially fractionated radiation treatment: Implications for endothelial apoptosis

“…different concentrations of human recombinant TNFα, which has been shown to be recognized by the murine TNFαRI but not TNFαRII. 31 Medium and cells were collected. The medium was cleared from dead cells by centrifugation at 80 x g, while the cells were homogenated in the buffer listed below.…”

Physiological and pathophysiological aspects of ceramide

“…In particular, it was demonstrated that ceramide inhibits the potassium channel Kv1.3 and calcium releaseactivated calcium channels in lymphocytes (32,67,100,151). Both channels are central for activation, differentiation, proliferation, and regulation of apoptosis.…”

Ischemia-Induced Exocytosis of Weibel-Palade Bodies Mobilizes Stem Cells

“…The tissue was minced and immediately homogenized, according to a previously published technique with minor modifications. 31 Cells were incubated for 30 min on ice with FITC-conjugated anti-mouse CD34 (RAM34; eBioscience, San Diego, CA) and PE-conjugated anti-mouse Flk-1 (Avas12␣1; BD Biosciences, Rockville, MD) or with FITC-conjugated anti-mouse CD117 (c-Kit; BD Biosciences) and PE-conjugated anti-mouse CD150 (eBioscience). After incubation, cells were washed with PBS and fixed in 4% paraformaldehyde.…”