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Nitric oxide synthase plays a signaling role in TCR-triggered apoptotic death
M S Williams1, S Noguchi, P A Henkart
1Department of Immunology, Holland Lab, American Red Cross, Rockville, MD 20855, USA. willmark@usa.redcross.org
Abstract:
A functional role for stimulated nitric oxide (NO) production was tested in the TCR-triggered death of mature T lymphocytes. In purified peripheral human T cell blasts or the 2B4 murine T cell hybridoma, apoptotic cell death induced by immobilized anti-CD3 was blocked by inhibitors of NO synthase (NOS) in a stereospecific and concentration-dependent manner. This effect appeared to be selective since apoptotic death induced by anti-Fas Ab or the steroid dexamethasone was not affected by NOS inhibitors. TCR-stimulated expression of functional Fas ligand was attenuated in a stereospecific manner by NOS inhibitors, but these compounds did not inhibit TCR-stimulated IL-2 secretion or CD69 surface expression. Nitrosylated tyrosines, a stable marker for NO generation, were immunochemically detected in T cells using flow cytometry. TCR signals induced NO production, as measured by an increase in nitrotyrosine-specific staining. NOS enzymatic activity was detected in lysates of 2B4 cells, and Western blot analysis suggests that the activity is due to expression of the neuronal isoform of NOS. Thus, T cells have the capacity to generate NO upon Ag signaling, which may affect signal transduction, Fas ligand surface expression, and apoptotic cell death of mature T lymphocytes.
Insights
Stimulated nitric oxide (NO) production plays a role in T cell death. Inhibiting NO synthase blocked T cell death and Fas ligand expression, indicating NO
Area of Science:
- Immunology
- Cell Biology
- Biochemistry
Background:
- T cell activation involves T cell receptor (TCR) signaling.
- Apoptosis, or programmed cell death, is crucial for immune system regulation.
- Nitric oxide (NO) is a signaling molecule with diverse biological functions.
Purpose of the Study:
- To investigate the role of nitric oxide (NO) production in T cell receptor (TCR)-triggered apoptosis of mature T lymphocytes.
- To determine if NO generation influences Fas ligand expression and cell death pathways.
Main Methods:
- Utilized purified human T cell blasts and a murine T cell hybridoma (2B4).
- Applied immobilized anti-CD3 antibodies to trigger TCR signaling and induce apoptosis.
- Employed nitric oxide synthase (NOS) inhibitors to assess the impact on cell death.
- Measured NO production via nitrotyrosine staining and NOS enzymatic activity assays.
- Analyzed Fas ligand expression, IL-2 secretion, and CD69 surface expression.
Main Results:
- Inhibitors of NO synthase (NOS) stereospecifically blocked TCR-induced apoptotic cell death.
- This blockade was selective, as NOS inhibitors did not affect apoptosis induced by anti-Fas antibodies or dexamethasone.
- TCR stimulation led to increased nitrotyrosine staining, indicating NO production.
- NOS inhibitors attenuated TCR-stimulated Fas ligand expression but not IL-2 secretion or CD69 expression.
- Western blot analysis suggested the presence of the neuronal isoform of NOS in T cells.
Conclusions:
- Mature T lymphocytes possess the capacity to generate nitric oxide (NO) upon antigen receptor (TCR) signaling.
- NO production influences TCR-mediated signal transduction, affecting Fas ligand surface expression.
- Nitric oxide plays a functional role in the apoptotic cell death of mature T lymphocytes.