T Cell Activation-Induced Mitochondrial Hyperpolarization Is Mediated by Ca2+- and Redox-Dependent Production of Nitric Oxide

Abstract

Activation, proliferation, or programmed cell death of T lymphocytes is regulated by the mitochondrial transmembrane potential (Δψm) through controlling ATP synthesis, production of reactive oxygen intermediates (ROI), and release of cell death-inducing factors. Elevation of Δψm or mitochondrial hyperpolarization is an early and reversible event associated with both T cell activation and apoptosis. In the present study, T cell activation signals leading to mitochondrial hyperpolarization were investigated. CD3/CD28 costimulation of human PBL elevated cytoplasmic and mitochondrial Ca2+ levels, ROI production, and NO production, and elicited mitochondrial hyperpolarization. Although T cell activation-induced Ca2+ release, ROI levels, and NO production were diminished by inositol 1,4,5-triphosphate receptor antagonist 2-aminoethoxydiphenyl borane, superoxide dismutase mimic manganese (III) tetrakis (4-benzoic acid) porphyrin chloride, spin trap 5-diisopropoxyphosphoryl-5-methyl-1-pyrroline-N-oxide, and NO chelator carboxy-2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide, mitochondrial hyperpolarization was selectively inhibited by carboxy-2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide (−85.0 ± 10.0%; p = 0.008) and, to a lesser extent, by 2-aminoethoxydiphenyl borane. Moreover, NO precursor (Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate diethylenetriamine elicited NO and ROI production, Ca2+ release, transient ATP depletion, and robust mitochondrial hyperpolarization (3.5 ± 0.8-fold; p = 0.002). Western blot analysis revealed expression of Ca-dependent endothelial NO synthase and neuronal NO synthase isoforms and absence of Ca-independent inducible NO synthase in PBL. CD3/CD28 costimulation or H2O2 elicited severalfold elevations of endothelial NO synthase and neuronal NO synthase expression, as compared with β-actin. H2O2 also led to moderate mitochondrial hyperpolarization; however, Ca2+ influx by ionomycin or Ca2+ release from intracellular stores by thapsigargin alone failed to induce NO synthase expression, NO production, or Δψm elevation. The results suggest that T cell activation-induced mitochondrial hyperpolarization is mediated by ROI- and Ca2+-dependent NO production.

Nitric oxide, mitochondrial hyperpolarization, and T cell activation☆

“…These observations were widely confirmed and extended to other apoptosis pathways [9,10,11,12,13,14]. Transient MHP is also triggered by activation of T cells by Con A [6] and CD3/CD28 costimulation [15] via ROI-and Ca 2+ -dependent production of NO [16]. Thus, MHP represents an early and reversible switch not exclusively associated with apoptosis.…”

“…NO chelator C-PTIO (carboxy-2-phenyl-4,4,5,5-tetrametyl-imidazoline-1-oxyl-3-oxide) profoundly inhibited TCR crosslinking induced mitochondrial hyperpolarization, cytoplasmic and mitochondrial Ca 2+ response, ROI production and NO levels, suggesting that TCR activation induced MHP is mediated by NO [16]. In addition, NO signal was required for CD3/CD28 induced Ca 2+ fluxing, suggesting an essential role of NO in T cell activation.…”

“…This is observed as a small and transient depolarization of the mitochondrial membrane in response in the early phase of the Ca 2+ flux. Although the activation of dehydrogenases stimulates mitochondrial respiration, leading to mitochondrial hyperpolarization (MHP) and increased ATP production, following the above mentioned initial depolarization in many cell types [1], Ca 2+ influx by ionomycin or Ca 2+ release from intracellular stores by thapsigargin alone failed to induce Δψ m elevation in lymphocytes [16].…”

“…Crosslinking of the TCR has been associated with elevation of the cytosolic Ca 2+ , ROI and nitric oxide (NO) production and mitochondrial hyperpolarization [16]. NO chelator C-PTIO (carboxy-2-phenyl-4,4,5,5-tetrametyl-imidazoline-1-oxyl-3-oxide) profoundly inhibited TCR crosslinking induced mitochondrial hyperpolarization, cytoplasmic and mitochondrial Ca 2+ response, ROI production and NO levels, suggesting that TCR activation induced MHP is mediated by NO [16].…”

“…These data suggest that principal mitochondrial functions are involved in T cell activation. Nitric oxide (NO) is produced by T cells during activation and it regulates T cell signal transduction [1,16,18]. NO affects both crucial functions of the mitochondrion, the generation of energy and the control of cell death through modulating production of reactive oxygen intermediates and of ATP.…”