Role of mitogen-activated protein kinases in activation-induced apoptosis of T cells

Abstract

A member of the mitogen-activated protein (MAP) kinase family, Jun N-terminal kinase (JNK), has been implicated in regulating apoptosis in various cell types. We have investigated the requirement for another type of MAP kinase, extracellular signal-regulated protein kinase (ERK) in activation-induced cell death (AICD) of T cells. AICD is the process by which recently activated T cells undergo apoptosis when restimulated through the T-cell antigen receptor. Here we show that both JNK and ERK are activated rapidly upon T-cell receptor (TCR) ligation prior to the onset of AICD. A chemical inhibitor of ERK activation, PD 098059, inhibits ERK activation and apoptosis, while JNK activation is not inhibited. This suggests that JNK activation is not sufficient for apoptosis. TCR cross-linking induces expression of the apoptosis-inducing factor, Fas ligand (FasL), and its expression correlates with ERK activation. In addition, apoptosis induced by direct ligation of the Fas receptor by anti-Fas antibody is not associated with ERK activation and is not inhibited by PD 098059. These data suggest that ERK activation is an early event during T-cell apoptosis induced by antigen-receptor ligation, and is not involved in apoptosis per se but in the expression of FasL. MAP kinase family members may be similarly involved in inducing apoptosis signals in other cell types.

Figure 1

Mitogen-activated protein (MAP) kinases are activated in Sup-T13 cells in response to anti-CD3 stimulation. Multivalent anti-CD3 monoclonal antibodies (mAbs) were obtained by incubating 5 μg/ml murine IgG2b antihuman CD3 mAb with 20 μg/ml goat antimouse IgG for 1 hr at room temperature. Sup-T13 cells were stimulated with or without multivalent anti-CD3 mAb for various times as indicated. For detection of ERK2 protein by Western blot (a), the blot was probed with anti-ERK2 rabbit IgG, followed by biotinylated goat antirabbit IgG and streptavidin-conjugated horseradish peroxidase. For detection of phosphorylated proteins, the blot was incubated with 1 μg/ml biotinylated anti-P-Tyr Ab, followed by streptavidin-conjugated horseradish peroxidase. The blots were detected by enhanced chemiluminescence. Molecular weight standards are indicated in the margin. This represents one of five experiments. The activities of ERK2 (b), JNK1 (c) and p38 (d) were measured by protein kinase assay, as described in the Materials and methods. A reaction containing no substrate was included as a negative control. Phorbol 12-myristate 13-acetate (PMA) (10 ng/ml) plus ionomycin (100 ng/ml) was used as a positive control to activate ERK2 at 2 min, JNK1 at 30 min and p38 at 4 hr. The kinase activity was measured in arbitrary units.

Figure 2

Anti-CD3-induced apoptosis of Sup-T13 cells is blocked by the MEK1 inhibitor PD 098059. Sup-T13 cells were cultured for 6–72 hr in medium alone, or with 40 μm PD 098059, plastic-bound anti-CD3 monoclonal antibody (mAb) or a combination of PD 098059 and anti-CD3 mAb. Cells were preincubated with PD 098059 for 30 min before culturing in anti-CD3-coated plastic plates. The percentage of DNA in each phase and the apoptotic DNA peak (Ap. Pk) were analysed by CELLQuest and MulticycleAV software.

Figure 3

PD 098059 inhibits anti-CD3 induced cell death in a time- and dose-dependent manner and selectively inhibits ERK activity. (a) Sup-T13 cells were preincubated with 40 μm PD 098059 for 30 min. PD 098059-treated cells were then cultured in medium alone or with plastic-bound anti-CD3 monoclonal antibody (mAb) for 24 hr, 48 hr or 72 hr. PD 098059 was not cytotoxic at this concentration. (b) Sup-T13 cells were stimulated with plastic-bound anti-CD3 mAb for 48 hr in the presence of various concentrations of PD 098059 or in medium alone. (c) PD 098059 specifically blocked ERK2, but not JNK1, activation in response to anti-CD3 or the combination of phorbol 12-myristate 13-acetate (PMA) and ionomycin. Cells were cultured with 40 μm PD 098059 or in medium alone for 30 min prior to the addition of multivalent anti-CD3 mAb or 10 ng/ml PMA plus 100 ng/ml ionomycin. The activities of ERK2 and JNK1 were measured in immunocomplex protein kinase assays using myelin basic protein (MBP) or GST-c-Jun as substrate. A reaction containing no substrate was also included as a negative control.

Figure 4

Extracellular signal-regulated protein kinase (ERK) activity is required in anti-CD3, but not in anti-Fas-induced apoptosis of antigen (Ag)-activated T cells from DO10 mice. To obtain Ag-activated T cells, splenocytes from T-cell receptor (TCR) transgenic DO10 mice were stimulated with 1 μm of ovalbumin (OVA) peptide for 3 days. Activated T cells were enriched by passage through a nylon wool column and cultured in fresh medium in the presence of 10 U/ml murine interleukin-2 (IL-2) for 1 day. Viable activated T cells were then harvested by Ficoll–Hypaque gradient centrifugation. (a) For kinase assay, anti-CD3 monoclonal antibody (mAb) or anti-Fas mAb at a concentration of 0·2 μg/ml and 2 μg/ml, respectively, were cross-linked by incubation with goat antihamster IgG at 10-fold excess for 1 hr at room temperature. Activated T cells (2 × 10⁷) were incubated with either multivalent anti-CD3 mAb or anti-Fas mAb for 0 min, 2 min, 30 min or 2 hr. ERK2 activity was assayed as described in the Materials and methods. (b) Cells were preincubated in medium alone or with 40 μm PD 098059 for 30 min. For anti-CD3 treatment, 2 × 10⁵ T cells were cultured in each well of a 96-well plate with medium alone, 0·2 μg/ml anti-CD3, 40 μm PD 098059 or a combination of anti-CD3 and PD 098059. For anti-Fas treatment, T cells were labelled with anti-Fas mAb for 30 min. Anti-Fas-labelled T cells at 2 × 10⁵/well were then seeded in the wells precoated with 20 μg/ml goat antihamster IgG in medium alone or in the presence of 40 μm PD 098059. Goat antihamster IgG treatment alone did not affect cell viability compared with medium alone (data not shown). All cultures contained 10 U/ml murine IL-2. Six hours after anti-CD3 or anti-Fas treatment, cells were harvested and nucleic DNA was stained and analysed with the CELLQuest program on flow cytometry.

Figure 5

Extracellular signal-regulated protein kinase (ERK) activity is required for the FasL expression of Sup-T13 cells in response to anti-CD3 stimulation. Three million Sup-T13 cells were preincubated in the presence of dimethylsulphoxide (DMSO; PD 098059 solvent) or 40 μm PD 098059 for 30 min. Cells were then added into six-well plates, precoated with or without 3 μg/ml anti-CD3 monoclonal antibody (mAb). The plate was spun at 150 g for 5 min. After 1, 2, 4 or 6 hr of incubation, cells were harvested. Total RNA was prepared and reverse transcribed to cDNA. Competitive reverse transcription–polymerase chain reaction (RT–PCR) was carried out as described in the Materials and methods. (a) An ethidium bromide-stained agarose gel shows two PCR products per lane representing discrete competitor (small fragment) and RT–cDNA (large fragment) bands for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or Fas ligand (FasL). In each sample, different ranges of competitor concentrations were used to permit analysis of unknown GAPDH or FasL message near the equivalence point, as described in the Materials and methods. The arrowheads indicate visual estimate of the equivalence point. The quantitative estimate of endogenous full-length cDNA is determined by linear regression of densitometrically analysed band intensity and is shown above each arrowhead. Sup-T13 cells were treated with either DMSO alone as a baseline control, or with 40 μm PD 098059 or 3 μg/ml anti-CD3 mAb, or with anti-CD3 mAb plus PD 098059, for 6 hr. The data represents one of four similar experiments. (b) The kinetics of FasL expression before and after anti-CD3 mAb stimulation in the presence or absence of PD 098059. Scanning densitometry of the competing bands enables a precise calculation of the data. Raw FasL gene-expression values were corrected by the amount of GAPDH RT–cDNA, as described in the Materials and methods.