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Discrete generation of superoxide and hydrogen peroxide by T cell receptor stimulation: selective regulation of mitogen-activated protein kinase activation and fas ligand expression.

Devadas S, Zaritskaya L, Rhee SG, Oberley L, Williams MS - J. Exp. Med. (2002)

Bottom Line: In this study, we have shown for the first time that TCR cross-linking induced rapid (within 15 min) generation of both hydrogen peroxide and superoxide anion, as defined with oxidation-sensitive dyes, selective pharmacologic antioxidants, and overexpression of specific antioxidant enzymes.Anti-CD3 induced phosphorylation of extracellular signal-regulated kinase (ERK)1/2 required hydrogen peroxide generation but was unaffected by superoxide anion.Thus, antigen receptor signaling induces generation of discrete species of oxidants that selectively regulate two distinct redox sensitive pathways, a proapoptotic (FasL) and a proliferative pathway (ERK).

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, Holland Laboratory, American Red Cross, Rockville, MD 20855, USA.

ABSTRACT
Receptor-stimulated generation of reactive oxygen species (ROS) has been shown to regulate signal transduction, and previous studies have suggested that T cell receptor (TCR) signals may involve or be sensitive to ROS. In this study, we have shown for the first time that TCR cross-linking induced rapid (within 15 min) generation of both hydrogen peroxide and superoxide anion, as defined with oxidation-sensitive dyes, selective pharmacologic antioxidants, and overexpression of specific antioxidant enzymes. Furthermore, the data suggest the novel observation that superoxide anion and hydrogen peroxide are produced separately by distinct TCR-stimulated pathways. Unexpectedly, TCR-stimulated activation of the Fas ligand (FasL) promoter and subsequent cell death was dependent upon superoxide anion, but independent of hydrogen peroxide, while nuclear factor of activated T cells (NFAT) activation or interleukin 2 transcription was independent of all ROS. Anti-CD3 induced phosphorylation of extracellular signal-regulated kinase (ERK)1/2 required hydrogen peroxide generation but was unaffected by superoxide anion. Thus, antigen receptor signaling induces generation of discrete species of oxidants that selectively regulate two distinct redox sensitive pathways, a proapoptotic (FasL) and a proliferative pathway (ERK).

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Anti-CD3–stimulated generation of ROS in 9C127 murine T cell hybridoma. (A) Representative FACS® profile for anti-CD3–induced DCFDA oxidation at 15 min with unstimulated cells (dashed lines) and anti-CD3–stimulated cells (filled profile). (B) Representative FACS® profile for anti-CD3 stimulated DHE oxidation at 60 min with unstimulated cells (dashed lines) and anti-CD3–stimulated cells (filled profile). (C) Kinetics of DCFDA/DHE oxidation in 9C127 cells. 9C127 cells were stimulated with anti-CD3 as described in Materials and Methods and the oxidation of DCFDA (○) and DHE (▪) was determined by FACS® analysis. The data are expressed as the percent stimulated increase in mean channel fluorescence of DCFDA/DHE over unstimulated controls and represent the average of at least five separate experiments (± SEM). *Anti-CD3–stimulated DCFDA oxidation is significantly different from unstimulated controls (P < 0.05). #Anti-CD3–stimulated DHE oxidation is significantly different from unstimulated controls (P < 0.05).
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fig1: Anti-CD3–stimulated generation of ROS in 9C127 murine T cell hybridoma. (A) Representative FACS® profile for anti-CD3–induced DCFDA oxidation at 15 min with unstimulated cells (dashed lines) and anti-CD3–stimulated cells (filled profile). (B) Representative FACS® profile for anti-CD3 stimulated DHE oxidation at 60 min with unstimulated cells (dashed lines) and anti-CD3–stimulated cells (filled profile). (C) Kinetics of DCFDA/DHE oxidation in 9C127 cells. 9C127 cells were stimulated with anti-CD3 as described in Materials and Methods and the oxidation of DCFDA (○) and DHE (▪) was determined by FACS® analysis. The data are expressed as the percent stimulated increase in mean channel fluorescence of DCFDA/DHE over unstimulated controls and represent the average of at least five separate experiments (± SEM). *Anti-CD3–stimulated DCFDA oxidation is significantly different from unstimulated controls (P < 0.05). #Anti-CD3–stimulated DHE oxidation is significantly different from unstimulated controls (P < 0.05).

Mentions: The kinetics of anti-CD3–stimulated ROS production in the murine T cell hybridoma 9C127 were measured using the cell permeant, oxidation sensitive dyes DCFDA or DHE. These dyes are nonfluorescent until oxidized by ROS and an increase in fluorescence of DCFDA (Fig. 1 A) indicates oxidation by peroxides, peroxynitrite, and/or hydroxyl radical (23), while DHE is selectively oxidized by superoxide anion (24) to the fluorescent product ethidium bromide (Fig. 1 B). Thus, DCFDA oxidation was measured as early as 15 min after TCR activation and was maintained throughout the time points monitored (Fig. 1 C). When these experiments were performed using preoxidized forms of DCFDA (fluorescein diacetate or dichlorofluorescein), anti-CD3 stimulation induced no alterations in fluorescence (data not shown) indicating that the observed changes in DCFDA signal were not due to altered uptake, sequestration, or de-esterification of the dye. Using DHE as a selective probe for superoxide anion, anti-CD3–stimulated oxidation of DHE was observed only at 45 and 60 min (Fig. 1 C). Thus, the data are consistent with anti-CD3–stimulated intracellular generation of distinct species of oxidants with different kinetics.


Discrete generation of superoxide and hydrogen peroxide by T cell receptor stimulation: selective regulation of mitogen-activated protein kinase activation and fas ligand expression.

Devadas S, Zaritskaya L, Rhee SG, Oberley L, Williams MS - J. Exp. Med. (2002)

Anti-CD3–stimulated generation of ROS in 9C127 murine T cell hybridoma. (A) Representative FACS® profile for anti-CD3–induced DCFDA oxidation at 15 min with unstimulated cells (dashed lines) and anti-CD3–stimulated cells (filled profile). (B) Representative FACS® profile for anti-CD3 stimulated DHE oxidation at 60 min with unstimulated cells (dashed lines) and anti-CD3–stimulated cells (filled profile). (C) Kinetics of DCFDA/DHE oxidation in 9C127 cells. 9C127 cells were stimulated with anti-CD3 as described in Materials and Methods and the oxidation of DCFDA (○) and DHE (▪) was determined by FACS® analysis. The data are expressed as the percent stimulated increase in mean channel fluorescence of DCFDA/DHE over unstimulated controls and represent the average of at least five separate experiments (± SEM). *Anti-CD3–stimulated DCFDA oxidation is significantly different from unstimulated controls (P < 0.05). #Anti-CD3–stimulated DHE oxidation is significantly different from unstimulated controls (P < 0.05).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2196010&req=5

fig1: Anti-CD3–stimulated generation of ROS in 9C127 murine T cell hybridoma. (A) Representative FACS® profile for anti-CD3–induced DCFDA oxidation at 15 min with unstimulated cells (dashed lines) and anti-CD3–stimulated cells (filled profile). (B) Representative FACS® profile for anti-CD3 stimulated DHE oxidation at 60 min with unstimulated cells (dashed lines) and anti-CD3–stimulated cells (filled profile). (C) Kinetics of DCFDA/DHE oxidation in 9C127 cells. 9C127 cells were stimulated with anti-CD3 as described in Materials and Methods and the oxidation of DCFDA (○) and DHE (▪) was determined by FACS® analysis. The data are expressed as the percent stimulated increase in mean channel fluorescence of DCFDA/DHE over unstimulated controls and represent the average of at least five separate experiments (± SEM). *Anti-CD3–stimulated DCFDA oxidation is significantly different from unstimulated controls (P < 0.05). #Anti-CD3–stimulated DHE oxidation is significantly different from unstimulated controls (P < 0.05).
Mentions: The kinetics of anti-CD3–stimulated ROS production in the murine T cell hybridoma 9C127 were measured using the cell permeant, oxidation sensitive dyes DCFDA or DHE. These dyes are nonfluorescent until oxidized by ROS and an increase in fluorescence of DCFDA (Fig. 1 A) indicates oxidation by peroxides, peroxynitrite, and/or hydroxyl radical (23), while DHE is selectively oxidized by superoxide anion (24) to the fluorescent product ethidium bromide (Fig. 1 B). Thus, DCFDA oxidation was measured as early as 15 min after TCR activation and was maintained throughout the time points monitored (Fig. 1 C). When these experiments were performed using preoxidized forms of DCFDA (fluorescein diacetate or dichlorofluorescein), anti-CD3 stimulation induced no alterations in fluorescence (data not shown) indicating that the observed changes in DCFDA signal were not due to altered uptake, sequestration, or de-esterification of the dye. Using DHE as a selective probe for superoxide anion, anti-CD3–stimulated oxidation of DHE was observed only at 45 and 60 min (Fig. 1 C). Thus, the data are consistent with anti-CD3–stimulated intracellular generation of distinct species of oxidants with different kinetics.

Bottom Line: In this study, we have shown for the first time that TCR cross-linking induced rapid (within 15 min) generation of both hydrogen peroxide and superoxide anion, as defined with oxidation-sensitive dyes, selective pharmacologic antioxidants, and overexpression of specific antioxidant enzymes.Anti-CD3 induced phosphorylation of extracellular signal-regulated kinase (ERK)1/2 required hydrogen peroxide generation but was unaffected by superoxide anion.Thus, antigen receptor signaling induces generation of discrete species of oxidants that selectively regulate two distinct redox sensitive pathways, a proapoptotic (FasL) and a proliferative pathway (ERK).

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, Holland Laboratory, American Red Cross, Rockville, MD 20855, USA.

ABSTRACT
Receptor-stimulated generation of reactive oxygen species (ROS) has been shown to regulate signal transduction, and previous studies have suggested that T cell receptor (TCR) signals may involve or be sensitive to ROS. In this study, we have shown for the first time that TCR cross-linking induced rapid (within 15 min) generation of both hydrogen peroxide and superoxide anion, as defined with oxidation-sensitive dyes, selective pharmacologic antioxidants, and overexpression of specific antioxidant enzymes. Furthermore, the data suggest the novel observation that superoxide anion and hydrogen peroxide are produced separately by distinct TCR-stimulated pathways. Unexpectedly, TCR-stimulated activation of the Fas ligand (FasL) promoter and subsequent cell death was dependent upon superoxide anion, but independent of hydrogen peroxide, while nuclear factor of activated T cells (NFAT) activation or interleukin 2 transcription was independent of all ROS. Anti-CD3 induced phosphorylation of extracellular signal-regulated kinase (ERK)1/2 required hydrogen peroxide generation but was unaffected by superoxide anion. Thus, antigen receptor signaling induces generation of discrete species of oxidants that selectively regulate two distinct redox sensitive pathways, a proapoptotic (FasL) and a proliferative pathway (ERK).

Show MeSH
Related in: MedlinePlus