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Expression of membrane-bound and soluble FasL in Fas- and FADD-dependent T lymphocyte apoptosis induced by mildly oxidized LDL¹

INSERM U466, Institut Louis Bugnard, CHU Rangueil and University Paul Sabatier, Toulouse, France

³Correspondence: INSERM U466, CHU Rangueil, 1 Avenue Jean Poulhès, TSA 50032 31059 Toulouse Cedex 9, France. E-mail:hbenoist{at}toulouse.inserm.fr

SPECIFIC AIMS

The present study was designed to investigate the role of Fas/FasL system in T lymphocyte apoptosis induced by mildly oxidized low density lipoproteins (oxLDL).

PRINCIPAL FINDINGS

1. oxLDL-induced apoptosis involves Fas/FasL system in PHA-activated T lymphocytes
Previously, we have reported that after 48 h exposure to 200 µg apoB/ml oxLDL 20–30% of phytohemagglutin (PHA)-activated peripheral blood mononuclear cells (PBMC) died by apoptosis. We now demonstrate that 200 µg/ml oxLDL increased Fas and FasL expression on PHA-activated T cells. Interestingly, the increase of FasL expression preceded phosphatidylserine externalization indicating that FasL translocation at the cell surface occurred before the apoptotic process. The loss of cell viability, DNA fragmentation and phosphatidylserine externalization were significantly inhibited by addition of a blocking anti-Fas antibody or a Fas-Fc chimeric fusion protein. Further, PHA-activated T cell enriched splenocytes from CEH-MRLlpr mice lacking functional Fas were strongly resistant to 100 µg/ml oxLDL and partially resistant to 200 µg/ml oxLDL. Altogether, our results indicate that oxLDL-induced apoptosis of T lymphocytes occurs mainly, but not entirely, through a Fas-dependent process.

2. oxLDL-induced Fas/FasL overexpression and apoptosis involve ROS production and MAP kinase activation in PHA-activated T lymphocytes
Using flow cytometry we showed that ROS production induced by PHA activation after 30 min incubation was increased 2.5 fold by oxLDL treatment (Fig. 1 A). In resting PBMC, oxLDL had no detectable effect on JNK activation and only slightly activated ERK and p38. In PHA-activated PBMC, oxLDL transiently enhanced ERK and JNK activation within the first hour of incubation (Fig. 1B ). PHA-activated PBMC were incubated with specific kinase inhibitors or anti-oxidant molecules and CD3⁺ lymphocytes were analyzed. Trolox and inhibitors of JNK or ERK clearly decreased the expression of both Fas and FasL at 24h, whereas Vitamin E partially inhibited FasL expression only (Fig. 1C ). JNK and ERK inhibitors as well as anti-oxidant molecules inhibited oxLDL-induced apoptosis after 48h of culture, as assessed by PS externalization in CD3⁺cells (Fig. 1D ). Altogether, our data indicate that an increase of ROS production, as well as ERK and JNK activation, are involved in oxLDL-induced Fas/FasL expression on activated T cells and in the resulting apoptosis.

View larger version (40K): [in this window] [in a new window]   Figure 1. Signaling pathways involved in oxLDL-induced Fas/FasL expression and apoptosis in PHA-actvated lymphocytes. A) PHA-activated PBMC were cultured for 30 min in the presence (thick histogram) or absence (thin histogram) of oxLDL (200 µg/ml). ROS production was measured in CD3+ lymphocytes by cytofluorimetric analysis. Histograms are representative of 4 independent experiments. B) PBMC were cultured for 30 min with or without PHA (1µg/ml) and oxLDL (200 µg/ml) as indicated. Expression of ERK, P38 and JNK was monitored by Western blotting. C) and D) PBMC were pre-incubated for an hour with or without specific kinase inhibitors (p38 inhibitor: SB203580 (10 µM), JNK II inhibitor: (5 µM), ERK inhibitor: PD098059 (5 µM)) or antioxidant molecules (Vitamin E (Vit E, 50 µM); Trolox (50 µM)) and further incubated with PHA (1 µg/ml) and oxLDL (200 µg/ml) for 24 or 48 h. Fas and FasL expression at 24h (C) and PS externalisation at 48h (D) were analyzed by flow cytometry on CD3+ lymphocytes. Data are expressed as the percentage of the value measured in PHA-activated PBMC incubated in the presence of oxLDL alone. Data are mean ± SD of 3 to 5 independent experiments.

3. oxLDL trigger FasL soluble secretion preceding Fas-dependent apoptosis of Jurkat T cell line
Previously, we demonstrated that after 24 h exposure to 200 µg apoB/ml oxLDL 50% of Jurkat cells died by apoptosis. Flow cytometry indicated that expression of Fas at the cell surface was up-regulated after 12 to 24 h of oxLDL treatment, in a dose dependent manner. In contrast to Fas, FasL was not constitutively expressed at the cell surface. FasL expression was evident after 24 h incubation with oxLDL. Soluble FasL (sFasL) was detected as early as 4 h in the presence of oxLDL and increased up to 351 ± 60 pg/ml (16 h) in a time dependent manner. In contrast, no TNF- was produced. Moreover, sFasL was released in the culture medium of 48h PHA-activated PBMC (325±45 pg/ml) and was potentiated by 200 µg apoB/ml oxLDL (758±65 pg/ml). Finally, the adding of a neutralizing anti-Fas antibody to oxLDL-treated Jurkat T cells reduced apoptosis. Thus, our results suggest that oxLDL-induced Jurkat T cell apoptosis occurs mainly through a Fas-dependent process and putatively involves soluble FasL production.

4. FADD protein is involved in oxLDL-mediated caspase activation and apoptosis of T cells
In T cells, Fas triggers caspase activation and apoptosis through a FADD-dependent pathway. After 24h incubation in presence of oxLDL, FADD-deficient Jurkat T-cells (FADD, Fig. 2 A) strongly resisted apoptosis as compared with Jurkat control cells (A3) (Fig. 2B ). OxLDL induced cleavage of caspases 8 and 3 already at 8 h in A3 cells. Caspase processing was completely impaired in FADD cells (Fig. 2C and D ). Altogether, these findings suggest that oxLDL induce caspase activation and apoptosis through a FADD-dependent pathway. Similarly, oxLDL-treatment of PHA-activated PBMC for 24 h induced activation of caspases 8 and 3 (Fig. 2E ), before significant signs of apoptosis appear.

View larger version (56K): [in this window] [in a new window]   Figure 2. Analysis of oxLDL-induced apoptosis in FADD-deficient Jurkat cells. A) Expression of FADD and ß-actin in control (A3) and FADD-deficient (FADD) Jurkat cells was visualized by Western blotting. B) control (A3) and FADD-deficient (FADD) Jurkat cells were incubated in the presence of the indicated amount of oxLDL. After 24 h, annexin V fluorescence was monitored by flow cytometry on propidium iodide-negative cells. C) Western blot showing caspase 8 activation in control (A3) and FADD-deficient (FADD) Jurkat cells after treatment with oxLDL (200 µg/ml) or FasL (20 ng/ml) for 8 h. D) kinetics of caspase 3 activation. E) for comparison is shown caspase 8 and 3 activation in PHA-activated PBMC cultured for 24h with or without various concentrations of oxLDL. Data from (B, C, D, and E) are representative of two independent experiments.

5. oxLDL increase intracellular ceramide in T lymphocytes
Ceramide, a putative second messenger of cell death, was reported to enhance Fas-induced Jurkat apoptosis triggered by soluble FasL. The ceramide level strongly increased in oxLDL-treated Jurkat cells but not in LDL-treated cells. Ceramide production started as early as 1 h and was maximal at 8 h. Interestingly, ceramide generation occurred before sFasL production and caspase activation. Moreover, oxLDL-induced ceramide production was not altered in FADD cells. In PHA-activated PBMC a rise of intracellular ceramide concentration was also observed in the presence of oxLDL and preceded a significant increase of apoptosis. Altogether, our results indicate that oxLDL-induced ceramide generation was neither a consequence of cell death nor a FADD-dependent process.

Conclusion and significance

The present study strongly suggests that mildly oxidized lipoproteins can induce Fas/FasL-mediated apoptosis of activated or proliferating T lymphocytes. Our data are in agreement with the concept that extensive LDL oxidation is not necessary to modulate cell responses and promote cell death.

We demonstrate for the first time that oxLDL can activate sFasL production in T lymphocytes. Because it has been previously demonstrated in vascular endothelial cells and in macrophages that oxLDL stimulate metalloproteinase activity, the present stimulation of sFasL production might be a consequence of metalloproteinase activation involved in membrane-bound FasL cleavage (Fig. 3 ).

View larger version (24K): [in this window] [in a new window]   Figure 3. Schematic diagram of the possible role of ROS, ERK, JNK, membrane-bound FasL, soluble FasL, and ceramide in apoptosis mediated by mildly oxidized LDL in activated or proliferating T lymphocytes. Solid lines: Mechanism proposed in previous studies, mainly using endothelial cells and smooth muscle cells, and supported by the present findings. Dotted lines: additional mechanisms supported by the present study.

SFasL is probably less cytotoxic than membrane-bound FasL, due to the incapacity of sFasL to induce effective capping of Fas. Recently, it was suggested that Fas capping is dependent on an increase of intracellular ceramide concentration. On the other hand, oxLDL can stimulate sphingomyelinase and ceramide generation. Our results in Jurkat T cells clearly indicate that oxLDL increase ceramide concentration and sFasL production before cell death occurs. Thus, oxLDL might potentiate the cell susceptibility to soluble FasL-mediated apoptosis by a rise of intracellular ceramide (Fig. 3) .

Previous works have demonstrated an involvement of ROS production, ERK and JNK in the regulation of FasL expression after T lymphocyte activation. Our data demonstrate, for the first time, that mildly oxidized LDL stimulate Fas and FasL expression in activated T lymphocytes through signaling pathways involving ROS production as well as ERK and JNK activation (Fig. 3) . Because oxLDL may stimulate various pathways of apoptosis simultaneously, oxLDL-mediated cell death is probably the result of the combination of different events: for instance in this work, Fas binding to membrane-bound FasL (cell interaction) and/or sFasL (autocrine or paracrine effect) associated with a concomitant increase of ceramide concentration (Fig. 3) .

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/1096/fj.02-0808fje;

² These authors contributed equally to this work.

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