Involvement of SLC17A9-dependent vesicular exocytosis in the mechanism of ATP release during T cell activation

Abstract

Recent reports have shown that T cell receptor (TCR)-dependent ATP release from T cells is involved in production of interleukin-2 (IL-2) through activation of P2 receptors. Stimulation of TCR induces ATP release from T cells through gap junction hemichannels and maxianion channels, at least in part. However, the mechanisms of ATP release from activated T cells are not fully understood. Here, we studied the mechanisms of ATP release during TCR-dependent T cell activation by investigating the effects of various inhibitors on TCR-dependent ATP release from murine T cells. We found that not only anion channel and gap junction hemichannel inhibitors, but also exocytosis inhibitors suppressed the ATP release. These results suggest that ATP release from murine T cells is regulated by various mechanisms, including exocytosis. An inhibitor of exocytosis, bafilomycin A, significantly blocked TCR signaling, such as Ca(2+) elevation and IL-2 production. Furthermore, bafilomycin A, ectonucleotidase, and P2Y(6) receptor antagonist significantly inhibited production of pro-inflammatory cytokines from external antigen-restimulated splenocytes, indicating that vesicular exocytosis-mediated purinergic signaling has a significant role in TCR-dependent cytokine production. We also detected vesicular ATP in murine T cells and human T lymphoma Jurkat cells, both of which also expressed mRNA of SLC17A9, a vesicular nucleotide transporter. Knockdown of SLC17A9 in Jurkat cells markedly reduced ATP release and cytosolic Ca(2+) elevation after TCR stimulation, suggesting involvement of SLC17A9-dependent vesicular exocytosis in ATP release and T cell activation. In conclusion, vesicular exocytosis of ATP appears to play a role in T cell activation and immune responses.

FIGURE 1.

Effects of pharmacological inhibition on TCR/CD28 stimulation-induced ATP release in murine T cells. A, splenocytes were stimulated by ligation of CD3/CD28 using Dynabeads and incubated for the indicated times. After incubation, the concentration of ATP in the culture medium was measured (n = 12–20). B, splenocytes were pretreated with carbenoxolone (CBX) (50 μm), 18-glycyrrhetinic acid (18GA) (50 μm), flufenamic acid (FFA) (50 μm), glibenclamide (100 μm), arachidonic acid (20 μm), GdCl₃ (100 μm), A438079 (100 μm), and bafilomycin A (50 nm) for 30 min. At 1 min after TCR/CD28 stimulation, the supernatants were collected and ATP content was measured (n = 12–54). Each value represents the mean ± S.E. A significant difference between the control group and the indicated group is represented (*, p < 0.05 or **, p < 0.01).

FIGURE 2.

Role of PI 3-kinase activation and elevation of intracellular Ca²⁺ in ATP release. A, splenocytes were pretreated with bafilomycin A (50 nm) and BFA (10 μm) for 30 min. At 1 min after TCR/CD28 stimulation, the supernatants were collected and ATP content was measured (n = 16–24). B, splenocytes were pretreated with LY294002 (10 μm) for 30 min. At 1 min after TCR/CD28 stimulation, the supernatants were collected and ATP content was measured (n = 6–10). C, splenocytes were pretreated with BAPTA-AM (50 μm) or incubated in Ca²⁺-free buffer for 30 min. At 1 min after TCR/CD28 stimulation, the supernatants were collected and ATP content was measured (n = 12–24). Each value represents the mean ± S.E. A significant difference between the control group and the indicated group is represented (**, p < 0.01).

FIGURE 3.

The effects of bafilomycin A on early events of murine T cell activation. A, after preincubation with bafilomycin A (50 nm) or vehicle for 30 min, splenocytes were stimulated with CD3/CD28 beads for 2 min in the absence or presence of bafilomycin A (50 nm) and stimulation was stopped by the addition of lysis buffer. Cell lysates were then subjected to immunoblotting with rabbit mAbs against phospho-Lck or Lck. B and C, after incubation of splenocytes with PE-Cy5 anti-CD4 mAb, splenocytes loaded with Fluo-4 were resuspended in Ca²⁺-free RPMI 1640-based buffer (B) or RPMI 1640-based buffer (C). Cells were stimulated with anti-CD3 and anti-CD28 mAbs, and then secondary Ab was added at 110 s after the first stimulation. The change in fluorescence was analyzed for a further 3 or 6 min. The cells were preincubated in the presence or absence of bafilomycin A (50 nm) for 15 min. We obtained qualitatively similar results in all experiments, and typical data from three independent experiments are presented.

FIGURE 4.

Effects of bafilomycin A on late events of murine T cell activation. A, splenocytes were incubated with plate-bound anti-CD3 and soluble anti-CD28 mAbs for 4 h in the presence or absence of bafilomycin A (50 nm), and then IL-2 mRNA levels were measured as described under “Experimental Procedures” (n = 3). B and C, splenocytes were incubated with plate-bound anti-CD3 and soluble anti-CD28 mAbs for 24 h in the presence or absence of bafilomycin A (50 nm), and then the concentration of IL-2 in culture medium (n = 10–14) (B) and the percentage of CD25⁺ cells in CD4⁺ T cells (n = 5–23) (C and D) were measured as described under “Experimental Procedures.” Each value represents the mean ± S.E. A significant difference between the control group (naive) and the indicated group is represented (**, p < 0.01).

FIGURE 5.

Suppression of cytokine secretion in mBSA-immunized mice. A–C, BALB/c mice were immunized with mBSA. Ten days after immunization, the spleen was harvested. Splenocytes (7.0 × 10⁶ cells/ml) were restimulated with mBSA in vitro and cultured with apyrase (20 units/ml), MRS2578 (10 μm), A438079 (100 μm), and bafilomycin A (50 nm) for 24 h. The supernatants were assayed for IL-2 (A), IL-6 (B), and IL-17 (C) (n = 8–10). Each value represents the mean ± S.E. A significant difference between control group (mBSA) and the indicated group is represented (**, p < 0.01).

FIGURE 6.

Existence of vesicular ATP and expression of mouse SLC17A9 mRNA in murine CD4 T cells. A, splenocytes were stained with MANT-ATP (50 μm) and anti-mouse CD4 mAb PE-Cy5 for 1 h at 37 °C. The fluorescence of MANT-ATP in CD4 T cells was analyzed using a confocal laser scanning microscope. B, expression levels of SLC17A9 mRNA in splenocytes and CD4 T cells were determined by RT-PCR. Expression of GAPDH mRNA is shown as a loading control.

FIGURE 7.

Involvement of vesicular exocytosis on TCR-dependent ATP release in Jurkat cells. A, Jurkat cells were stimulated by ligation of CD3/CD28 using Dynabeads and incubated for the indicated times. After incubation, the concentration of ATP in the culture medium was measured (n = 14–16). B, splenocytes were pretreated with bafilomycin A (50 nm) or BFA (10 μm) for 30 min. At 1 min after TCR/CD28 stimulation, the supernatants were collected and ATP content was measured (n = 6–16). C, Jurkat cells were stained with MANT-ATP (50 μm) for 1 h at 37 °C. Stained cells were analyzed using a confocal laser scanning microscope. D, expression levels of human SLC17A9 mRNA in Jurkat cells were determined by RT-PCR. Expression of GAPDH is shown as a loading control. Each value represents the mean ± S.E. A significant difference between control group and the indicated group is represented (**, p < 0.01).

FIGURE 8.

Involvement of SLC17A9 in ATP release in TCR-dependent ATP release in Jurkat cells. A, Jurkat cells were transfected with shRNA targeting SLC17A9 or the negative control shRNA, and gene expression of SLC17A9 was examined by assessing mRNA levels using real time RT-PCR (n = 3). B, Jurkat cells transfected with shRNA targeting SLC17A9 or the negative control shRNA were stimulated by ligation of CD3/CD28 using Dynabeads. At 1 min after TCR/CD28 stimulation, the supernatants were collected and ATP content was measured (n = 8–12). C, Jurkat cells loaded with Fluo-4 were stimulated with anti-CD3 mAbs. The cells were preincubated in the presence or absence of bafilomycin A (50 nm) for 15 min. D, the shRNA-transfected cells loaded with Fluo-4 were stimulated with anti-CD3 mAbs. The change in fluorescence was measured for 7 min. Each value represents the mean ± S.E. A significant difference between the control group (scramble) and the indicated group is represented (*, p < 0.05 and **, p < 0.01).