Stimulation of CD95 (Fas) blocks T lymphocyte calcium channels through sphingomyelinase and sphingolipids

Abstract

Calcium influx through store-operated calcium release-activated calcium channels (CRAC) is required for T cell activation, cytokine synthesis, and proliferation. The CD95 (Apo-1͞Fas) receptor plays a role in self-tolerance and tumor immune escape, and it mediates apoptosis in activated T cells. In this paper we show that CD95-stimulation blocks CRAC and Ca 2؉ influx in lymphocytes through the activation of acidic sphingomyelinase (ASM) and ceramide release. The block of Ca 2؉ entry is lacking in CD95-defective lpr lymphocytes as well as in ASM-defective cells and can be restored by retransfection of ASM. C2 ceramide, C6 ceramide, and sphingosine block CRAC reversibly, whereas the inactive dihydroceramide has no effect. CD95-stimulation or the addition of ceramide prevents store-operated Ca 2؉ The CD95-receptor induces apoptosis in lymphocytes and is critically involved in the maintenance of self-tolerance (reviewed in ref. 5). Expression of the CD95 ligand (CD95L) can create immune privileged sites (6, 7), and malignant tumors may escape from the immune attack by expressing high levels of CD95L (8,9).However, CD95 may have additional functions apart from lymphocyte apoptosis. First, freshly activated T lymphocytes express CD95 but are resistant to CD95-mediated apoptosis (10, 11). Second, in tumor patients, defective lymphocyte function rather than apoptotic deletion has been observed (12, 13). Third, mice inoculated with a CD95L-expressing tumor develop severe CD95-mediated defects in lymphocyte function (14). Fourth, the knockout of CD95-activated signaling molecules required for apoptosis does not, in contrast to CD95-knockout, result in lymphoproliferative disease (15,16). Thus, apoptosis may not represent the only function of CD95 in immune tolerance. Materials and MethodsCells. Jurkat T cells, Niemann-Pick Disease type A (NPDA), Daudi, and JY B cells were maintained in RPMI medium 1640, and LN18 and LN229 glioblastoma were maintained in DMEM (17). The stimulating monoclonal mouse anti-human CD95 (CH11; Upstate Biotechnology, Lake Placid, NY) was added at 100 ng͞ml. Apoptosis was assayed by nuclear fragmentation (Hoe33342, propidium iodide or SYTO 16; Molecular Probes) or FITC-annexin flow cytometry. Peripheral T lymphoblasts were prepared by using a Ficoll gradient, purified with nylon wool columns (Ͼ90% CD3 ϩ ), and stimulated with 1 g͞ml phythaemaglutinin (PHA) (24 h) plus 2.5 units͞ml IL-2 (4-6 days). Thymocytes were prepared from homozygous C57͞Bl6 lpr-and gld mice (ages 10-15 weeks). Transient transfection of NPDA cells was performed by electroporation with an expression vector encoding acidic sphingomyelinase (ASM) (pJK-asm, 15 g͞ml) and a Myc-tagged single chain (18). Twelve hours after electroporation viable cells were purified by Ficoll gradient centrifugation and cultured for an additional 24 h. After 36 h, Myc presented on the cell surface was stained with a monoclonal anti-Myc-antibody (9E10; American Type Culture Collection, Bethesda, MD) and a phycoerythrin-conjugated secondary antibody (18...

Involvement of Sphingosine in Mitochondria-dependent Fas-induced Apoptosis of Type II Jurkat T Cells

“…The results presented here may reconcile many findings, as we observed a biphasic ceramide response to Fas ligation in type II Jurkat cells as follows: a rapid and transient low amplitude increase in the cellular concentration of ceramide followed by a delayed, persistent, and robust elevation. Thus, these data are consistent with the reports in which a rapid rise in ceramide has been implicated as an early event in the Fas-signaling pathway in Jurkat cells (10,14,20,21), as well as association solely with a late increase in ceramide (15,27). It is important to note that in many other cell types in which apoptosis occurs more slowly than in Jurkat T cells, such as MCF-7 human breast cancer cells in response to TNF␣ (46,73), the "early" rise in ceramide occurs at a later time, albeit still prior to the effector phase of apoptosis in these cells.…”

“…However, we have found that exogenous C 2 -ceramide also increases levels of sphingosine as well as endogenous ceramide in Jurkat cells. Rapid generation of endogenous ceramide in cells treated with C 2 -ceramide has also been previously reported in Jurkat cells (21) and U937 cells (48). The early increases of ceramide and sphingosine after treatment with C 2 -ceramide are similar to those achieved after Fas ligation in Jurkat cells.…”

“…Followed by a Sustained Accumulation of Ceramide-As previously reported (10,14,20,21), we found that treatment of Jurkat cells with 50 ng/ml anti-Fas antibody resulted in acute changes in ceramide levels within 30 min from a basal level of ϳ10 pmol/nmol total phospholipids (Fig. 1A).…”

Inhibition of the Calcium Release-activated Calcium (CRAC) Current in Jurkat T Cells by the HIV-1 Envelope Protein gp160

“…The (42), sphingosine and several ceramides in Jurkat T cells (43), and PKC in mast cells (44) and Jurkat T cells (39). A PKC-dependent process also seems to be involved in the reduction of I CRAC by gp160.…”

A Functionalized Sphingolipid Analogue for Studying Redistribution during Activation in Living T Cells

“…Similarly, physiological acid SMase (ASM) activation after death receptor ligation followed by ceramide release interferes with relay of TCR signaling by abrogating store-operated Ca 2+ entry, NF-AT activation, and IL-2 synthesis (25,26). However, in other situations, activation of SMases appears to modulate T cell activation (27)(28)(29), indicating that both the amount and/or compartmentalization of ceramides at stimulatory interfaces needs to be tightly controlled.…”