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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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Functional role for superoxide in anti-CD3–stimulated cell death. (A) 9C127 cells were unstimulated (hatched bars) or incubated on immobilized anti-CD3 (solid bars) for 16 h in the presence or absence of the SOD mimetic MnTBaP. Cells were harvested and apoptosis (hypodiploid cells) was measured after staining with propidium iodide by flow cytometry as described in Materials and Methods. The data are expressed as the percent apoptotic cells and represent the average of three separate experiments ± SEM. *Significantly different from untreated controls (P < 0.002). (B and C) 9C127 cells were transfected with empty vector (Vector) or an expression vector encoding CuZnSOD or TPx at a 2:1 ratio with one encoding GFP as a transfection marker. After 16 h culture, cells were incubated for 24 h further on immobilized anti-CD3 (solid bars) or in the absence of stimulation (hatched bars). The percent total viable (PI−) cells (B) and those expressing GFP (C) were quantitated by flow cytometry. Anti-CD3–stimulated death of productively transfected (GFP+) viable cells for vector transfected cells was 58.7 ± 6.3%, while that in CuZnSOD-transfected cells was 21.1 ± 7.4% (significantly different from vector control; P < 0.05) and in TPx overexpressing cells it was 39.7 ± 4.3% (not significantly different from vector control; P > 0.2). The data represent the average of three separate experiments ± SEM. *Significantly different from unstimulated controls (P < 0.01); #Significantly different from unstimulated controls (P < 0.05); †Not significantly different from unstimulated controls (P > 0.1).
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fig6: Functional role for superoxide in anti-CD3–stimulated cell death. (A) 9C127 cells were unstimulated (hatched bars) or incubated on immobilized anti-CD3 (solid bars) for 16 h in the presence or absence of the SOD mimetic MnTBaP. Cells were harvested and apoptosis (hypodiploid cells) was measured after staining with propidium iodide by flow cytometry as described in Materials and Methods. The data are expressed as the percent apoptotic cells and represent the average of three separate experiments ± SEM. *Significantly different from untreated controls (P < 0.002). (B and C) 9C127 cells were transfected with empty vector (Vector) or an expression vector encoding CuZnSOD or TPx at a 2:1 ratio with one encoding GFP as a transfection marker. After 16 h culture, cells were incubated for 24 h further on immobilized anti-CD3 (solid bars) or in the absence of stimulation (hatched bars). The percent total viable (PI−) cells (B) and those expressing GFP (C) were quantitated by flow cytometry. Anti-CD3–stimulated death of productively transfected (GFP+) viable cells for vector transfected cells was 58.7 ± 6.3%, while that in CuZnSOD-transfected cells was 21.1 ± 7.4% (significantly different from vector control; P < 0.05) and in TPx overexpressing cells it was 39.7 ± 4.3% (not significantly different from vector control; P > 0.2). The data represent the average of three separate experiments ± SEM. *Significantly different from unstimulated controls (P < 0.01); #Significantly different from unstimulated controls (P < 0.05); †Not significantly different from unstimulated controls (P > 0.1).

Mentions: To test the significance of this selective role of ROS, the functional role of superoxide anion in anti-CD3–stimulated apoptosis and cell death was determined. Previous studies using antioxidants had suggested that ROS generation was required for anti-CD3–stimulated apoptosis of mature T cells (11). Incubation with the SOD mimetic, MnTBaP, at a concentration (50 μM) that blocked DHE oxidation, almost completely blocked anti-CD3–stimulated apoptosis of 9C127 cells (Fig. 6 A). This suggests that superoxide anion is necessary for AICD, but as MnTBaP also has peroxidase activity (Fig. 2), additional experiments in cells overexpressing CuZnSOD were performed to solidify a role for superoxide. 9C127 cells were cotransfected with CuZnSOD and GFP as above and after 16 h incubation to allow protein expression the cells were stimulated for 24 h on immobilized anti-CD3. As loss of membrane integrity (cell death) will result in loss of GFP within the cell, the expression of GFP was used as an indicator of whether overexpression of a cotransfected protein would lead to a survival advantage. In vector-transfected cells, anti-CD3 stimulation led to ∼70% death of total cells (Fig. 6 B) or 60% of those cells detected as GFP+ (Fig. 6 C). The same data were obtained when an irrelevant protein (β-galactosidase) was overexpressed (not shown). In cells transfected with CuZnSOD, anti-CD3–induced death of the total cell population was similar to that in the vector transfected samples. However, the stimulated death of those cells productively transfected with CuZnSOD (GFP+ cells) was significantly inhibited (only 21% cell death; Fig. 6 C), suggesting that cells overexpressing CuZnSOD were selectively resistant to anti-CD3–induced cell death. These data suggest that the inhibition of FasL expression by SOD overexpression is also reflected by an inhibition of anti-CD3–induced cell death. Overexpression of TPx also induced a slight decrease in the death of GFP+ cells (Fig. 6 C), although it was not significantly different from the death of vector transfected cells.


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)

Functional role for superoxide in anti-CD3–stimulated cell death. (A) 9C127 cells were unstimulated (hatched bars) or incubated on immobilized anti-CD3 (solid bars) for 16 h in the presence or absence of the SOD mimetic MnTBaP. Cells were harvested and apoptosis (hypodiploid cells) was measured after staining with propidium iodide by flow cytometry as described in Materials and Methods. The data are expressed as the percent apoptotic cells and represent the average of three separate experiments ± SEM. *Significantly different from untreated controls (P < 0.002). (B and C) 9C127 cells were transfected with empty vector (Vector) or an expression vector encoding CuZnSOD or TPx at a 2:1 ratio with one encoding GFP as a transfection marker. After 16 h culture, cells were incubated for 24 h further on immobilized anti-CD3 (solid bars) or in the absence of stimulation (hatched bars). The percent total viable (PI−) cells (B) and those expressing GFP (C) were quantitated by flow cytometry. Anti-CD3–stimulated death of productively transfected (GFP+) viable cells for vector transfected cells was 58.7 ± 6.3%, while that in CuZnSOD-transfected cells was 21.1 ± 7.4% (significantly different from vector control; P < 0.05) and in TPx overexpressing cells it was 39.7 ± 4.3% (not significantly different from vector control; P > 0.2). The data represent the average of three separate experiments ± SEM. *Significantly different from unstimulated controls (P < 0.01); #Significantly different from unstimulated controls (P < 0.05); †Not significantly different from unstimulated controls (P > 0.1).
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fig6: Functional role for superoxide in anti-CD3–stimulated cell death. (A) 9C127 cells were unstimulated (hatched bars) or incubated on immobilized anti-CD3 (solid bars) for 16 h in the presence or absence of the SOD mimetic MnTBaP. Cells were harvested and apoptosis (hypodiploid cells) was measured after staining with propidium iodide by flow cytometry as described in Materials and Methods. The data are expressed as the percent apoptotic cells and represent the average of three separate experiments ± SEM. *Significantly different from untreated controls (P < 0.002). (B and C) 9C127 cells were transfected with empty vector (Vector) or an expression vector encoding CuZnSOD or TPx at a 2:1 ratio with one encoding GFP as a transfection marker. After 16 h culture, cells were incubated for 24 h further on immobilized anti-CD3 (solid bars) or in the absence of stimulation (hatched bars). The percent total viable (PI−) cells (B) and those expressing GFP (C) were quantitated by flow cytometry. Anti-CD3–stimulated death of productively transfected (GFP+) viable cells for vector transfected cells was 58.7 ± 6.3%, while that in CuZnSOD-transfected cells was 21.1 ± 7.4% (significantly different from vector control; P < 0.05) and in TPx overexpressing cells it was 39.7 ± 4.3% (not significantly different from vector control; P > 0.2). The data represent the average of three separate experiments ± SEM. *Significantly different from unstimulated controls (P < 0.01); #Significantly different from unstimulated controls (P < 0.05); †Not significantly different from unstimulated controls (P > 0.1).
Mentions: To test the significance of this selective role of ROS, the functional role of superoxide anion in anti-CD3–stimulated apoptosis and cell death was determined. Previous studies using antioxidants had suggested that ROS generation was required for anti-CD3–stimulated apoptosis of mature T cells (11). Incubation with the SOD mimetic, MnTBaP, at a concentration (50 μM) that blocked DHE oxidation, almost completely blocked anti-CD3–stimulated apoptosis of 9C127 cells (Fig. 6 A). This suggests that superoxide anion is necessary for AICD, but as MnTBaP also has peroxidase activity (Fig. 2), additional experiments in cells overexpressing CuZnSOD were performed to solidify a role for superoxide. 9C127 cells were cotransfected with CuZnSOD and GFP as above and after 16 h incubation to allow protein expression the cells were stimulated for 24 h on immobilized anti-CD3. As loss of membrane integrity (cell death) will result in loss of GFP within the cell, the expression of GFP was used as an indicator of whether overexpression of a cotransfected protein would lead to a survival advantage. In vector-transfected cells, anti-CD3 stimulation led to ∼70% death of total cells (Fig. 6 B) or 60% of those cells detected as GFP+ (Fig. 6 C). The same data were obtained when an irrelevant protein (β-galactosidase) was overexpressed (not shown). In cells transfected with CuZnSOD, anti-CD3–induced death of the total cell population was similar to that in the vector transfected samples. However, the stimulated death of those cells productively transfected with CuZnSOD (GFP+ cells) was significantly inhibited (only 21% cell death; Fig. 6 C), suggesting that cells overexpressing CuZnSOD were selectively resistant to anti-CD3–induced cell death. These data suggest that the inhibition of FasL expression by SOD overexpression is also reflected by an inhibition of anti-CD3–induced cell death. Overexpression of TPx also induced a slight decrease in the death of GFP+ cells (Fig. 6 C), although it was not significantly different from the death of vector transfected cells.

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