Escherichia coli enterotoxin B subunit triggers apoptosis of CD8(+) T cells by activating transcription factor c-myc

Abstract

Heat-labile enterotoxin from enterotoxinogenic Escherichia coli is not only an important cause of diarrhea in humans and domestic animals but also possesses potent immunomodulatory properties. Recently, the nontoxic, receptor-binding B subunit of heat-labile enterotoxin (EtxB) was found to induce the selective death of CD8(+) T cells, suggesting that EtxB may trigger activation of proapoptotic signaling pathways. Here we show that EtxB treatment of CD8(+) T cells but not of CD4(+) T cells triggers the specific up-regulation of the transcription factor c-myc, implicated in the control of cell proliferation, differentiation, and death. A concomitant elevation in Myc protein levels was also evident, with peak expression occurring 4 h posttreatment. Preincubation with c-myc antisense oligodeoxynucleotides demonstrated that Myc expression was necessary for EtxB-mediated apoptosis. Myc activation was also associated with an increase of IkappaBalpha turnover, suggesting that elevated Myc expression may be dependent on NF-kappaB. When CD8(+) T cells were pretreated with inhibitors of IkappaBalpha turnover and NF-kappaB translocation, this resulted in a marked reduction in both EtxB-induced apoptosis and Myc expression. Further, a non-receptor-binding mutant of EtxB, EtxB(G33D), was shown to lack the capacity to activate Myc transcription. These findings provide further evidence that EtxB is a signaling molecule that triggers activation of transcription factors involved in cell survival.

FIG. 1

EtxB induces c-myc gene expression in CD8⁺ T cells. Purified CD8⁺ (a) and CD4⁺ (b) T cells derived from mesenteric lymph nodes were incubated for 6 h in the presence of PBS, 30 μg of EtxB/ml, or 30 μg of EtxB(G33D)/ml. Total RNA was extracted and hybridized with a labeled myc multiprobe template set as described in Materials and Methods. Unprotected probes, mouse control RNA, and yeast tRNA were also run simultaneously with the samples. Identical results were obtained on three independent occasions. Quantification of c-myc gene expression in CD8⁺ (c) and CD4⁺ (d) T cells incubated for 4 and 6 h in the presence of PBS (white columns), 30 μg of EtxB/ml (black columns), and 30 μg of EtxB(G33D)/ml (grey columns) was obtained by normalizing c-myc band intensity in comparison with the GAPDH housekeeping gene. Mean values ± standard deviations of three independent experiments are shown.

FIG. 1

EtxB induces c-myc gene expression in CD8⁺ T cells. Purified CD8⁺ (a) and CD4⁺ (b) T cells derived from mesenteric lymph nodes were incubated for 6 h in the presence of PBS, 30 μg of EtxB/ml, or 30 μg of EtxB(G33D)/ml. Total RNA was extracted and hybridized with a labeled myc multiprobe template set as described in Materials and Methods. Unprotected probes, mouse control RNA, and yeast tRNA were also run simultaneously with the samples. Identical results were obtained on three independent occasions. Quantification of c-myc gene expression in CD8⁺ (c) and CD4⁺ (d) T cells incubated for 4 and 6 h in the presence of PBS (white columns), 30 μg of EtxB/ml (black columns), and 30 μg of EtxB(G33D)/ml (grey columns) was obtained by normalizing c-myc band intensity in comparison with the GAPDH housekeeping gene. Mean values ± standard deviations of three independent experiments are shown.

FIG. 2

EtxB induces c-myc protein overexpression and IκBα degradation in CD8⁺ T cells. (a) CD8⁺ and CD4⁺ mesenteric T cells were incubated for 4 and 6 h in the presence of PBS and 30 μg of EtxB/ml. −, absence of EtxB; +, presence of EtxB. The Myc level is denoted on the left in kilodaltons. (b) Total IκBα degrades faster in CD8⁺ T cells treated with EtxB. CD8⁺ T cells were incubated with cycloheximide (20 μg/ml) in the presence or absence of 30 μg of EtxB/ml, and the reaction was terminated at the time points shown. IκBα level is denoted on the left in kilodaltons. (c) SN50 blocks EtxB-induced c-myc protein expression in CD8⁺ T cells. CD8⁺ T cells were preincubated for 2 h with 50 μg of SN50/ml and were then treated for a further 6 h in the presence of 30 μg of EtxB/ml. Myc and IκBα protein levels were detected by Western blotting as described in Materials and Methods. The Myc level is denoted on the left in kilodaltons. Identical results were obtained in three independent experiments.

FIG. 3

Myc AS-ODNs reduce EtxB-induced apoptosis in CD8⁺ T cells. CD8⁺ T cells were incubated with PBS (a) or EtxB (b). (c) EtxB-treated cells were also preincubated for 4 h with 10 μM Myc AS-ODNs. Cells were then stained with propidium iodide, and the proportion of CD8⁺ T cells in the apoptotic subdiploid (G₀/G₁) stage was determined by flow cytometry analysis. The percentage of total subdiploid cells is indicated on top of the respective peak. (d) CD8⁺ T cells were sham treated or preincubated for 4 h with 10 μM Myc AS-ODNs in the presence or absence of 30 μg of EtxB/ml, and the reaction was terminated at 6 h. Myc protein levels were detected as described in Materials and Methods and measured in kilodaltons, as marked on the left. The results shown in the figure are typical of three independent experiments.

FIG. 4

Inhibition of IκBα turnover by TPCK drastically reduces EtxB-induced apoptosis in CD8⁺ T cells. CD8⁺ T cells were incubated with PBS (a) or 30 μg of EtxB/ml (b). Cells were also preincubated for 2 h with 25 μM TPCK and were subsequently treated with EtxB (c) for a further 24 h. Propidium iodide staining and flow cytometry analysis were performed as for Fig. 3. (d) TPCK reduces IκBα turnover in EtxB-treated CD8⁺ T cells. CD8⁺ T cells were incubated with cycloheximide (20 μg/ml) in the presence of 30 μg of EtxB/ml alone or EtxB plus 25 μM TPCK for 2 and 4 h. IκBα protein levels were detected by Western blotting as for Fig. 2b and are denoted on the left in kilodaltons. Identical results were obtained in three independent experiments.

FIG. 5

TPCK down-regulates c-myc expression in CD8⁺ T cells. CD8⁺ and CD4⁺ T cells were preincubated for 2 h in the presence of 25 μM TPCK and were then treated with 30 μg of EtxB/ml for a further 6 h. Total RNA was extracted and hybridized with a labeled myc multiprobe template set. Quantification of c-myc, Max, and Mad1 gene expression in CD8⁺ (a) and CD4⁺ (b) T cells treated with PBS (white columns), EtxB (black columns), or a combination of TPCK and EtxB (striped columns) was obtained by normalizing c-myc, Max, and Mad1 expression to L32 housekeeping gene expression. The results are mean values ± standard deviations of three independent experiments.