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Characterization of ionotropic glutamate receptors in human lymphocytes
G Lombardi1, C Dianzani, G Miglio
1DISCAFF Department, University of Piemonte Orientale, 28100 Novara, Italy. lombardi@pharm.unipmn.it
Insights
L-glutamate (Glu) enhances human lymphocyte activation by potentiating calcium ion rises, but inhibits proliferation at higher concentrations. This suggests lymphocytes express functional ionotropic glutamate receptors that modulate immune cell activity.
Area of Science:
- Immunology
- Neuroscience
- Cell Biology
Background:
- L-glutamate (Glu) is a primary excitatory neurotransmitter in the central nervous system.
- Its role in peripheral immune cells, particularly human lymphocytes, is less understood.
- Investigating Glu's impact on lymphocyte function is crucial for understanding neuro-immune interactions.
Purpose of the Study:
- To investigate the effect of L-glutamate (Glu) on human lymphocyte function.
- To determine if human lymphocytes express functional ionotropic glutamate receptors.
- To analyze Glu's influence on lymphocyte activation and proliferation.
Main Methods:
- Measurement of intracellular calcium ion ([Ca(2+)](i)) rise using the Fura-2 method.
- Assessment of cell proliferation via the MTT assay.
- Stimulation of lymphocytes with anti-CD(3) monoclonal antibody (mAb) or phytohaemagglutinin (PHA).
Main Results:
- L-glutamate (Glu) significantly potentiated anti-CD(3) mAb or PHA-induced intracellular Ca(2+) ([Ca(2+)](i)) rises in human lymphocytes.
- The potentiating effect of Glu on [Ca(2+)](i) showed a bell-shaped concentration-dependent relationship, peaking at 1 microM.
- Non-NMDA receptor agonists demonstrated higher efficacy than NMDA or Glu itself in potentiating [Ca(2+)](i) rises.
- Ionotropic Glu receptor antagonists, including NBQX and KYNA, effectively inhibited Glu-mediated effects.
- While Glu did not affect resting lymphocyte proliferation, it significantly inhibited PHA-induced proliferation at 1 mM.
Conclusions:
- Human lymphocytes express functional ionotropic glutamate receptors.
- These receptors modulate lymphocyte activation, primarily by influencing calcium signaling.
- Glu acts as a modulator of human lymphocyte activation, with concentration-dependent effects on proliferation.
Abstract:
The effect of L-glutamate (Glu) on human lymphocyte function was studied by measuring anti-CD(3) monoclonal antibody (mAb) or phytohaemagglutinin (PHA)-induced intracellular Ca(2+) ([Ca(2+)](i)) rise (Fura-2 method), and cell proliferation (MTT assay). Glu (0.001 - 100 microM) did not modify basal lymphocyte [Ca(2+)](i), but significantly potentiated the effects of anti-CD(3) mAb or PHA. Maximal [Ca(2+)](i) rises over resting cells were: 165+/-8 and 247+/-10 nM at 3.0x10(-2) mg ml(-1) anti-CD(3) mAb; 201+/-4 and 266+/-9 nM at 5.0x10(-2) mg ml(-1) PHA, in the absence or presence of 1 microM Glu, respectively. The Glu effect showed a bell-shape concentration-dependent relationship, with a maximum (+90+/-3% for anti-CD(3) mAb and +57+/-2% for PHA over Glu-untreated cells) at 1 microM. Non-NMDA receptor agonists (1 microM) showed a greater efficacy (+76+/-2% for (S)-AMPA; +78+/-4% for KA), if compared to NMDA (+46+/-2%), or Glu itself. Ionotropic Glu receptor antagonists completely inhibited the effects of the corresponding specific receptor agonists (1 microM). The IC(50) values calculated were: 0.9 microM for D-AP5; 0.6 microM for (+)-MK801; 0.3 microM for NBQX. Both NBQX and KYNA were able to abolish Glu effect. The IC(50s) calculated were: 3.4 microM for NBQX; 0.4 microM for KYNA. Glu (0.1 - 1 mM) did not change the resting cell proliferation, whereas Glu (1 mM) significant inhibited (-27+/-4%) PHA (1.0x10(-2) mg ml(-1))-induced lymphocyte proliferation at 72 h. In conclusion, human lymphocytes express ionotropic Glu receptors functionally operating as modulators of cell activation.