Group I mGlu receptor stimulation inhibits activation-induced cell death of human T lymphocytes

Abstract

1. The effects of L-glutamate on activation-induced cell death (AICD) of human activated (1 microg ml(-1) phytohemagglutinin plus 2 U ml(-1) interleukin-2; 8 days) T lymphocytes were studied by measuring anti-CD3 monoclonal antibody (10 microg ml(-1); 18 h)-induced cell apoptosis (Annexin V and propidium iodide staining). 2. L-Glutamate (1 x 10(-8)-1 x 10(-4) M) significantly (P < or = 0.01) inhibited AICD in a concentration-dependent manner (EC50=6.3 x 10(-8) M; maximum inhibition 54.8+/-6.3% at 1 x 10(-6) M). 3. The L-glutamate inhibitory effect was pharmacologically characterized as mediated by group I mGlu receptors, since mGlu receptor agonists reproduced this effect. The EC50 values were: 3.2 x 10(-7) M for (1S,3R)-ACPD; 4.5 x 10(-8) M for quisqualate; 1.0 x 10(-6) M for (S)-3,5-DHPG; 2.0 x 10(-5) M for CHPG. 4. Group I mGlu receptor antagonists inhibited the effects of quisqualate 1.0 x 10(-6) M. The IC50 values calculated were: 8.7 x 10(-5), 4.3 x 10(-6) and 6.3 x 10(-7) M for AIDA, LY 367385 and MPEP, respectively. 5. L-Glutamate (1 x 10(-6) M; 18 h) significantly (P < or = 0.05) inhibited FasL expression (40.8+/-11.3%) (cytofluorimetric analysis), whereas it did not affect Fas signalling. 6. Expression of both mGlu1 and mGlu5 receptor mRNA by T lymphocytes and T-cell lines, as demonstrated by reverse transcriptase-PCR analysis, suggests that L-glutamate-mediated inhibition of AICD was exerted on T cells. 7. These data depict a novel role for L-glutamate in the regulation of the immune response through group I mGlu receptor-mediated mechanisms.

Figure 1

Concentration–response curves for the L-glutamate or iGlu/mGlu receptor agonists inhibitory effects on AICD of human T lymphocytes. Activated T cells were stimulated with anti-CD3 mAb (10 μg ml⁻¹; 18 h) (a); in the presence of increasing concentrations (1 × 10⁻⁸–1 × 10⁻³ M) of L-glutamate, NMDA, AMPA or kainate (b), (1S,3R)-ACPD, quisqualate, L-CGG-I or L-AP4 (c), (S)-3,5-DHPG or CHPG (d), and the surviving cells were counted by cytofluorimetric analysis after staining with Annexin V plus propidium iodide. CD3 stimulation induced a 44.5±2.2% of AICD in comparison to anti-CD3-untreated cells. The EC₅₀ values were: 6.3 × 10⁻⁸ M for L-glutamate, 3.2 × 10⁻⁷ M for (1S,3R)-ACPD, 4.5 × 10⁻⁸ M for quisqualate, 1.0 × 10⁻⁶ M for (S)-3,5-DHPG and 2.0 × 10⁻⁵ M for CHPG. The white and dashed bars show the relative cell loss in anti-CD3-untreated and -treated cells in the absence of other drugs (a). The results are expressed as the mean±s.e.m. of at least ten experiments. ^*P⩽0.05; ^**P⩽0.01 versus anti-CD3 mAb-treated cells.

Figure 2

Inhibition of the L-glutamate, (1S,3R)-ACPD or quisqualate protective effects on AICD of human T lymphocytes. Activated T cells were stimulated with anti-CD3 mAb (10 μg ml⁻¹; 18 h) in the presence of (1 × 10⁻⁶ M) L-glutamate (a), (1 × 10⁻⁴ M) (1S,3R)-ACPD (b), (1 × 10⁻⁶ M) quisqualate (c) and increasing concentrations (1 × 10⁻⁸–1 × 10⁻³ M) of AIDA, LY 367385 or MPEP. Surviving cells were counted by cytofluorimetric analysis after staining with Annexin V plus propidium iodide. The IC₅₀ values, calculated when quisqualate was used, were: 8.7 × 10⁻⁵ M for AIDA; 4.3 × 10⁻⁶ M for LY 367385 and 6.3 × 10⁻⁷ M for MPEP. Dot lines represent the relative cell loss in anti-CD3 mAb-treated cells in the presence of L-glutamate (1 × 10⁻⁶ M) (a), (1S,3R)-ACPD (1 × 10⁻⁴ M) (b) quisqualate (1 × 10⁻⁶ M) (c). The results are expressed as the mean±s.e.m. of cell death of at least eight experiments. ^*P⩽0.05, ^**P⩽0.01 versus anti-CD3 mAb-treated cells in the presence of mGlu receptor agonists.

Figure 3

FasL expression in human T lymphocytes. Activated T cells were stimulated with anti-CD3 mAb (10 μg ml⁻¹; 18 h), in the presence of GM6001 (3 × 10⁻⁵ M), and in the absence or presence of L-glutamate (1 × 10⁻⁶ M). FasL expression evaluated by direct immunofluorescence and cytofluorimetric analysis using a fluorescein isothiocyanate (FITC)-conjugated anti-FasL mAb (a). (b) FasL expression of L-glutamate (1 × 10⁻⁶ M) untreated or treated T cells expressed as MFI-R (see Methods) of total T lymphocytes. Activated T cells were stimulated with anti-CD3 mAb (10 μg ml⁻¹; 18 h) in the presence of GM6001 (3 × 10⁻⁵ M) and in the absence or presence of L-glutamate (1 × 10⁻⁶ M). Surviving cells were counted by cytofluorimetric analysis after staining with Annexin V plus propidium iodide (c). The results are expressed as the mean±s.e.m. of cell death of at least four experiments. ^*P⩽0.05, ^**P⩽0.01 versus anti-CD3 mAb-treated cells in the absence of L-glutamate.

Figure 4

Fas-induced T-cell death. Activated T cells were stimulated with anti-Fas mAb (1 μg ml⁻¹; 18 h), in the absence or presence of L-glutamate (1 × 10⁻⁶ M). Surviving cells were counted by cytofluorimetric analysis after staining with Annexin V plus propidium iodide. The results are expressed as the mean±s.e.m. of cell death of at least four experiments.

Figure 5

Expression of mGlu₁ and mGlu₅ receptors in human lymphoid and myeloid cells. RT–PCRs were performed on total RNA isolated from mouse fibroblasts L(tk⁻) cells stably transfected with human mGlu₁ or mGlu₅ receptor cDNA (C+), FRO, SUP-T1, H9, HuT-78, Jurkat, THP-1 cell lines, human MDM, human resting T cell (R-CD3⁺) or human PHA (1 μg ml⁻¹) plus IL-2 (2 U ml⁻¹)-activated T cells (A-CD3⁺) using specific primers; in the negative control (C-) reverse trascriptase was omitted. GAPDH was used as house keeping gene to normalize cDNA amount. The results are representative of at least five experiments.