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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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Effect of ROS scavengers and chemical inhibitors of ROS. Anti-CD3–induced ROS generation was determined as described in Materials and Methods. DCFDA oxidation (○) was determined after 15 min and DHE oxidation (▪) after 60 min stimulation with anti-CD3 in the presence or absence of titrated concentrations of (A) MnTBaP, (B) ebselen, (C) BHA, (D) diphenylene iodonium (DPI), or (E) BAPTA-AM. The data are normalized to anti-CD3–induced DCFDA/DHE oxidation in the absence of drug and are expressed as percentage change in anti-CD3–stimulated fluorescence compared with cells exposed to drug alone. The data represent the average of at least three experiments (± SEM). *Anti-CD3–stimulated DCFDA oxidation is significantly different from that in the absence of drug (P < 0.05). #Anti-CD3–stimulated DHE oxidation is significantly different from that in the absence of drug (P < 0.05).
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fig2: Effect of ROS scavengers and chemical inhibitors of ROS. Anti-CD3–induced ROS generation was determined as described in Materials and Methods. DCFDA oxidation (○) was determined after 15 min and DHE oxidation (▪) after 60 min stimulation with anti-CD3 in the presence or absence of titrated concentrations of (A) MnTBaP, (B) ebselen, (C) BHA, (D) diphenylene iodonium (DPI), or (E) BAPTA-AM. The data are normalized to anti-CD3–induced DCFDA/DHE oxidation in the absence of drug and are expressed as percentage change in anti-CD3–stimulated fluorescence compared with cells exposed to drug alone. The data represent the average of at least three experiments (± SEM). *Anti-CD3–stimulated DCFDA oxidation is significantly different from that in the absence of drug (P < 0.05). #Anti-CD3–stimulated DHE oxidation is significantly different from that in the absence of drug (P < 0.05).

Mentions: To further characterize the ROS generated upon TCR stimulation in 9C127 cells, antioxidants were used as scavengers of selective species of oxidants. In all experiments DCFDA oxidation was measured 15 min after anti-CD3 cross-linking, while DHE oxidation was determined at 60 min. The SOD mimetic MnTBaP, which has been shown to possess both SOD and peroxidase activity in vitro (25), inhibited oxidation of both DCFDA and DHE in a dose-dependent manner (Fig. 2 A). In contrast, the hydroxyl radical scavenger BHA (Fig. 2 B), and the glutathione peroxidase (GPx) mimetic ebselen (Fig. 2 C) only inhibited DCFDA oxidation while having a limited effect on TCR-stimulated DHE oxidation. The data suggest that hydrogen peroxide or some derivative thereof (as detected by DCFDA oxidation) is rapidly produced after TCR activation and that the early hydrogen peroxide generation is not required for the later generation of superoxide anion (as measured by DHE oxidation).


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)

Effect of ROS scavengers and chemical inhibitors of ROS. Anti-CD3–induced ROS generation was determined as described in Materials and Methods. DCFDA oxidation (○) was determined after 15 min and DHE oxidation (▪) after 60 min stimulation with anti-CD3 in the presence or absence of titrated concentrations of (A) MnTBaP, (B) ebselen, (C) BHA, (D) diphenylene iodonium (DPI), or (E) BAPTA-AM. The data are normalized to anti-CD3–induced DCFDA/DHE oxidation in the absence of drug and are expressed as percentage change in anti-CD3–stimulated fluorescence compared with cells exposed to drug alone. The data represent the average of at least three experiments (± SEM). *Anti-CD3–stimulated DCFDA oxidation is significantly different from that in the absence of drug (P < 0.05). #Anti-CD3–stimulated DHE oxidation is significantly different from that in the absence of drug (P < 0.05).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196010&req=5

fig2: Effect of ROS scavengers and chemical inhibitors of ROS. Anti-CD3–induced ROS generation was determined as described in Materials and Methods. DCFDA oxidation (○) was determined after 15 min and DHE oxidation (▪) after 60 min stimulation with anti-CD3 in the presence or absence of titrated concentrations of (A) MnTBaP, (B) ebselen, (C) BHA, (D) diphenylene iodonium (DPI), or (E) BAPTA-AM. The data are normalized to anti-CD3–induced DCFDA/DHE oxidation in the absence of drug and are expressed as percentage change in anti-CD3–stimulated fluorescence compared with cells exposed to drug alone. The data represent the average of at least three experiments (± SEM). *Anti-CD3–stimulated DCFDA oxidation is significantly different from that in the absence of drug (P < 0.05). #Anti-CD3–stimulated DHE oxidation is significantly different from that in the absence of drug (P < 0.05).
Mentions: To further characterize the ROS generated upon TCR stimulation in 9C127 cells, antioxidants were used as scavengers of selective species of oxidants. In all experiments DCFDA oxidation was measured 15 min after anti-CD3 cross-linking, while DHE oxidation was determined at 60 min. The SOD mimetic MnTBaP, which has been shown to possess both SOD and peroxidase activity in vitro (25), inhibited oxidation of both DCFDA and DHE in a dose-dependent manner (Fig. 2 A). In contrast, the hydroxyl radical scavenger BHA (Fig. 2 B), and the glutathione peroxidase (GPx) mimetic ebselen (Fig. 2 C) only inhibited DCFDA oxidation while having a limited effect on TCR-stimulated DHE oxidation. The data suggest that hydrogen peroxide or some derivative thereof (as detected by DCFDA oxidation) is rapidly produced after TCR activation and that the early hydrogen peroxide generation is not required for the later generation of superoxide anion (as measured by DHE oxidation).

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