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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 protein overexpression on anti-CD3–induced reporter plasmid activation. (A) 9C127 cells (solid bars) or human T blasts (hatched bars) were transfected with empty vector (Vector) or expression vectors encoding BCl-xL, catalase, CuZnSOD, MnSOD, or TPx at a 2:1 ratio with one containing a 0.5 kb portion of the FasL promoter inserted into a luciferase reporter vector. (B) 9C127 cells were transfected with the same vectors at a 2:1 ratio with luciferase reporter vectors containing the minimal IL-2 promoter (hatched bars) and a multimerized NFAT binding site (open bars). Anti-CD3–stimulated luciferase activity in cell lysates was measured by chemoluminescence as described in Materials and Methods. Data are expressed as fold induction of luciferase activity over unstimulated and represents the average of at least four experiments. *Significantly different from Vector transfected controls (P < 0.05).
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fig5: Effect of protein overexpression on anti-CD3–induced reporter plasmid activation. (A) 9C127 cells (solid bars) or human T blasts (hatched bars) were transfected with empty vector (Vector) or expression vectors encoding BCl-xL, catalase, CuZnSOD, MnSOD, or TPx at a 2:1 ratio with one containing a 0.5 kb portion of the FasL promoter inserted into a luciferase reporter vector. (B) 9C127 cells were transfected with the same vectors at a 2:1 ratio with luciferase reporter vectors containing the minimal IL-2 promoter (hatched bars) and a multimerized NFAT binding site (open bars). Anti-CD3–stimulated luciferase activity in cell lysates was measured by chemoluminescence as described in Materials and Methods. Data are expressed as fold induction of luciferase activity over unstimulated and represents the average of at least four experiments. *Significantly different from Vector transfected controls (P < 0.05).

Mentions: Overexpression of antioxidant enzymes in human T blasts was also used to characterize the species of oxidants generated upon anti-CD3 stimulation. Overexpression of MnSOD and CuZnSOD both completely blocked anti-CD3–induced DHE oxidation, while catalase and TPx had little or no inhibitory effect (Fig. 5 B). These data are consistent with that observed in 9C127 cells and are supported by the observation that BCl-xL also completely inhibited TCR-stimulated DHE oxidation. Western blot analysis of transfected cells indicated that there was significant overexpression of proteins in productively transfected cells, which were purified based upon expression of the transfection marker (Fig. 4 C).


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 protein overexpression on anti-CD3–induced reporter plasmid activation. (A) 9C127 cells (solid bars) or human T blasts (hatched bars) were transfected with empty vector (Vector) or expression vectors encoding BCl-xL, catalase, CuZnSOD, MnSOD, or TPx at a 2:1 ratio with one containing a 0.5 kb portion of the FasL promoter inserted into a luciferase reporter vector. (B) 9C127 cells were transfected with the same vectors at a 2:1 ratio with luciferase reporter vectors containing the minimal IL-2 promoter (hatched bars) and a multimerized NFAT binding site (open bars). Anti-CD3–stimulated luciferase activity in cell lysates was measured by chemoluminescence as described in Materials and Methods. Data are expressed as fold induction of luciferase activity over unstimulated and represents the average of at least four experiments. *Significantly different from Vector transfected controls (P < 0.05).
© Copyright Policy
Related In: Results  -  Collection

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fig5: Effect of protein overexpression on anti-CD3–induced reporter plasmid activation. (A) 9C127 cells (solid bars) or human T blasts (hatched bars) were transfected with empty vector (Vector) or expression vectors encoding BCl-xL, catalase, CuZnSOD, MnSOD, or TPx at a 2:1 ratio with one containing a 0.5 kb portion of the FasL promoter inserted into a luciferase reporter vector. (B) 9C127 cells were transfected with the same vectors at a 2:1 ratio with luciferase reporter vectors containing the minimal IL-2 promoter (hatched bars) and a multimerized NFAT binding site (open bars). Anti-CD3–stimulated luciferase activity in cell lysates was measured by chemoluminescence as described in Materials and Methods. Data are expressed as fold induction of luciferase activity over unstimulated and represents the average of at least four experiments. *Significantly different from Vector transfected controls (P < 0.05).
Mentions: Overexpression of antioxidant enzymes in human T blasts was also used to characterize the species of oxidants generated upon anti-CD3 stimulation. Overexpression of MnSOD and CuZnSOD both completely blocked anti-CD3–induced DHE oxidation, while catalase and TPx had little or no inhibitory effect (Fig. 5 B). These data are consistent with that observed in 9C127 cells and are supported by the observation that BCl-xL also completely inhibited TCR-stimulated DHE oxidation. Western blot analysis of transfected cells indicated that there was significant overexpression of proteins in productively transfected cells, which were purified based upon expression of the transfection marker (Fig. 4 C).

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