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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–stimulated DHE oxidation in 9C127 cells. 9C127 cells were transfected with the indicated vectors at a 2:1 ratio with one encoding GFP as a transfection marker. After 16 h incubation, anti-CD3–induced DHE oxidation was measured as described in Materials and Methods. (A) “Normal sized” cells as defined by R1 in the FSC/SSC plot were analyzed. (B) Representative GFP staining profile. GFP expressing, productively transfected cells were defined by R2. (C) Relationship of GFP fluorescence to basal DHE staining indicating that expression of GFP did not affect dye uptake. (D) Representative DHE profile of “normal sized,” GFP+ cells with unstimulated cells (dashed lines) and anti-CD3–stimulated cells (filled profile). (E) Effect of protein overexpression on anti-CD3–induced DHE oxidation in 9C127 cells. DHE oxidation was determined after 60 min anti-CD3 stimulation in “normal sized,” GFP+ cells and was normalized to Vector control. The data represent the average of at least four separate experiments (± SEM). #Significantly different from that measured in Vector transfected samples (P < 0.05).
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fig3: Effect of protein overexpression on anti-CD3–stimulated DHE oxidation in 9C127 cells. 9C127 cells were transfected with the indicated vectors at a 2:1 ratio with one encoding GFP as a transfection marker. After 16 h incubation, anti-CD3–induced DHE oxidation was measured as described in Materials and Methods. (A) “Normal sized” cells as defined by R1 in the FSC/SSC plot were analyzed. (B) Representative GFP staining profile. GFP expressing, productively transfected cells were defined by R2. (C) Relationship of GFP fluorescence to basal DHE staining indicating that expression of GFP did not affect dye uptake. (D) Representative DHE profile of “normal sized,” GFP+ cells with unstimulated cells (dashed lines) and anti-CD3–stimulated cells (filled profile). (E) Effect of protein overexpression on anti-CD3–induced DHE oxidation in 9C127 cells. DHE oxidation was determined after 60 min anti-CD3 stimulation in “normal sized,” GFP+ cells and was normalized to Vector control. The data represent the average of at least four separate experiments (± SEM). #Significantly different from that measured in Vector transfected samples (P < 0.05).

Mentions: 9C127 T cell hybridoma or human T blasts (40 × 106/ml) were transiently transfected by electroporation (Gene Pulser; Bio-Rad Laboratories). Expression plasmids for BCl-xL, catalase, CuZnSOD, N17Rac1, MnSOD, TPx, or an empty vector (Vector Laboratories) were cotransfected with vectors encoding either GFP or RFP in a 2:1 ratio to ensure that any cell expressing GFP or RFP would also express the protein of interest. After 16 h incubation, transfected cells were harvested, counted, and assayed for anti-CD3–stimulated ROS production as described above. GFP fluorescence was used as a marker to gate on transfected cells in experiments using DHE while RFP fluorescence was used to gate on transfected cells in experiments with DCFDA. As shown in Fig. 3 C, expression of fluorescent proteins did not affect dye uptake.


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–stimulated DHE oxidation in 9C127 cells. 9C127 cells were transfected with the indicated vectors at a 2:1 ratio with one encoding GFP as a transfection marker. After 16 h incubation, anti-CD3–induced DHE oxidation was measured as described in Materials and Methods. (A) “Normal sized” cells as defined by R1 in the FSC/SSC plot were analyzed. (B) Representative GFP staining profile. GFP expressing, productively transfected cells were defined by R2. (C) Relationship of GFP fluorescence to basal DHE staining indicating that expression of GFP did not affect dye uptake. (D) Representative DHE profile of “normal sized,” GFP+ cells with unstimulated cells (dashed lines) and anti-CD3–stimulated cells (filled profile). (E) Effect of protein overexpression on anti-CD3–induced DHE oxidation in 9C127 cells. DHE oxidation was determined after 60 min anti-CD3 stimulation in “normal sized,” GFP+ cells and was normalized to Vector control. The data represent the average of at least four separate experiments (± SEM). #Significantly different from that measured in Vector transfected samples (P < 0.05).
© Copyright Policy
Related In: Results  -  Collection

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

fig3: Effect of protein overexpression on anti-CD3–stimulated DHE oxidation in 9C127 cells. 9C127 cells were transfected with the indicated vectors at a 2:1 ratio with one encoding GFP as a transfection marker. After 16 h incubation, anti-CD3–induced DHE oxidation was measured as described in Materials and Methods. (A) “Normal sized” cells as defined by R1 in the FSC/SSC plot were analyzed. (B) Representative GFP staining profile. GFP expressing, productively transfected cells were defined by R2. (C) Relationship of GFP fluorescence to basal DHE staining indicating that expression of GFP did not affect dye uptake. (D) Representative DHE profile of “normal sized,” GFP+ cells with unstimulated cells (dashed lines) and anti-CD3–stimulated cells (filled profile). (E) Effect of protein overexpression on anti-CD3–induced DHE oxidation in 9C127 cells. DHE oxidation was determined after 60 min anti-CD3 stimulation in “normal sized,” GFP+ cells and was normalized to Vector control. The data represent the average of at least four separate experiments (± SEM). #Significantly different from that measured in Vector transfected samples (P < 0.05).
Mentions: 9C127 T cell hybridoma or human T blasts (40 × 106/ml) were transiently transfected by electroporation (Gene Pulser; Bio-Rad Laboratories). Expression plasmids for BCl-xL, catalase, CuZnSOD, N17Rac1, MnSOD, TPx, or an empty vector (Vector Laboratories) were cotransfected with vectors encoding either GFP or RFP in a 2:1 ratio to ensure that any cell expressing GFP or RFP would also express the protein of interest. After 16 h incubation, transfected cells were harvested, counted, and assayed for anti-CD3–stimulated ROS production as described above. GFP fluorescence was used as a marker to gate on transfected cells in experiments using DHE while RFP fluorescence was used to gate on transfected cells in experiments with DCFDA. As shown in Fig. 3 C, expression of fluorescent proteins did not affect dye uptake.

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